Psma binding agents and uses thereof

ABSTRACT

The present invention relates to antibodies specifically binding PSMA or PSMA and CD3, polynucleotides encoding the antibodies or fragments, and methods of making and using the foregoing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser. No. 62/676,099, filed 24 May 2018. The entire content of the aforementioned application is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 30, 2019, is named PRD3342USNP1_SL.txt and is 269,122 bytes in size.

TECHNICAL FIELD

The disclosure provided herein relates to monoclonal antibodies that immunospecifically bind Prostate specific membrane antigen (PSMA), multispecific antibodies that immunospecifically bind PSMA and cluster of differentiation 3 (CD3), and methods of producing and using the described antibodies.

BACKGROUND

Prostate cancer is the second most common cancer in men worldwide, and the sixth leading cause of cancer-related death. Globally, there are approximately 1,100,000 new cases and 300,000 mortalities every year, comprising 4 percent of all cancer deaths. It is estimated that 1 in every 6 men will be diagnosed with the disease during his lifetime. In the U.S., more than 90% of prostate cancers are found in local or regional stages. At these early stages, the 5-year survival rate nears 100%. When the cancer has metastasized, however, the 5-year survival rate drops to 28%, and there remains a need for effective treatments for advanced-stage prostate cancer.

Prostate specific membrane antigen (PSMA), is a type II membrane protein that is highly expressed in prostatic intraepithelial neoplasia (PIN), a condition in which some prostate cells have begun to look and behave abnormally, and in primary and metastatic prostate cancers [Bostwick D G, Pacelli A, Blute M, Roche P, Murphy G P. Prostate specific membrane antigen expression in prostatic intraepithelial neoplasia and adenocarcinoma: A study of 184 cases. Cancer 1998; 82 (11):2256-2261.]. Expression of PSMA in cancer tissues correlates with the stage of disease and Gleason score [Kawakami M, Nakayama J. Enhanced expression of prostate-specific membrane antigen gene in prostate cancer as revealed by in situ hybridization. Cancer Res 1997; 57(12):2321-2324.]. PSMA expression is also higher in prostate cancer cells from hormone-refractory patients [Wright G L Jr, Grob B M, Haley C, Grossman K, Newhall K, Petrylak D, Troyer J, KonchubaA, Schellhammer P F, Moriarty R. Upregulation of prostate-specific membrane antigen after androgen-deprivation therapy. Urology 1996; 48(2):326-334.] and increased PSMA expression has been shown to be an independent marker of disease recurrence [Mitsiades C S, Lembessis P, Sourla A, Milathianakis C, TsintavisA, Koutsilieris M. Molecular staging by RT-pCR analysis for PSA and PSMA in peripheral blood and bone marrow samples is an independent predictor of time to biochemical failure following radical prostatectomy for clinically localized prostate cancer. Clin Exp Metastasis 2004; 21(6):495-505.]. High-level PSMA expression is correlated with early prostate-specific antigen (PSA) recurrence in surgically treated prostate cancer. PSMA expression levels correlate with the aggressiveness of the disease, and thereby strongly support PSMA as an excellent target for prostate cancer characterization and subsequent therapy.

Current treatments for prostate cancer include surgery, radiation and hormone therapies. When prostate cancers grow despite the lowering of testosterone levels by hormone therapy, treatment options are limited. Typically, the cancer vaccine sipuleucel-T, a radiopharmaceutical agent (such as radium-223 chloride), secondary hormone therapies (such as abiraterone or enzalutamide), and/or chemotherapies (docetaxel and cabazitaxel) are added to the hormonal therapy in sequence. While each of these treatments can delay growth of the cancer for several months and palliate symptoms produced by the disease, the disease ultimately becomes resistant to them. This underscores the need for more improved treatment and effective therapies for PSMA-expressing advanced prostate cancer.

SUMMARY

Provided herein are antibodies that immunospecifically bind to Pan Iroglodyles (chimpanzee, chimp), Macaca fascicularis (cynomolgus monkey, macaque, cyno) and/or human, PSMA and antigen-binding fragments thereof. Also described are related polynucleotides capable of encoding the provided PSMA-specific antibodies and antigen-binding fragments, cells expressing the provided antibodies and antigen-binding fragments, as well as associated vectors and detectably labeled antibodies and antigen-binding fragments. In addition, methods of using the provided antibodies and antigen-binding fragments are described. For example, the PSMA-specific antibodies and antigen-binding fragments may be used to diagnose or monitor PSMA-expressing cancer progression, regression, or stability; to determine whether or not a patient should be treated for cancer; or to determine whether or not a subject is afflicted with PSMA-expressing cancer and thus may be amenable to treatment with a PSMA-specific anti-cancer therapeutic, such as the multispecific (bispecific, trispecific, etc) antibodies against PSMA and CD3 described herein.

Further provided herein are multispecific antibodies that immunospecifically bind to PSMA and CD3 and multispecific antigen-binding fragments thereof. Also described are related polynucleotides capable of encoding the provided PSMA×CD3-multispecific antibodies, cells expressing the provided antibodies, as well as associated vectors and detectably labeled multispecific antibodies. In addition, methods of using the provided multispecific antibodies are described. For example, the PSMA×CD3-multispecific antibodies may be used to diagnose or monitor PSMA-expressing cancer progression, regression, or stability; to determine whether or not a patient should be treated for cancer; or to determine whether or not a subject is afflicted with PSMA-expressing cancer and thus may be amenable to treatment with a PSMA-specific anti-cancer therapeutic, such as the PSMA×CD3-multispecific antibodies described herein.

PSMA-Specific Antibodies

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis (cynomolgus monkey, macaque, cyno) and/or human PSMA, comprising a heavy chain complementarity determining region 1 (HCDR1), a HCDR2 and a HCDR3 of SEQ ID NOs: 31, 42 and 43, respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs: 31, 42, 43, respectively, and a light chain complementarity determining region 1 (LCDR1), a LCDR2 and a LCDR3 of SEQ ID NOs: 11, 12 and 13 respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs: 25, 26 and 27, respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs: 25, 26, 27, respectively, and a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs: 28, 29 and 30 respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes. Macaca fascicularis and/or human PSMA, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs: 20, 21 and 22, respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs: 20, 21, and 22, respectively, and a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs: 23, 12 and 24 respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs: 14, 15 and 16, respectively, or SEQ ID NOs: 14, 15 and 16 respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs: 14, 15, and 16, respectively, and a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs: 17, 18 and 19 respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs: 36, 37 and 38, respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes. Macaca fascicularis and/or human PSMA, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs: 36, 37, and 38, respectively, and a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs: 39, 40 and 41 respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs: 122, 123 and 124, respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs: 122, 123, and 124, respectively, and a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs: 23, 12, and 24, respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis (cynomolgus monkey, macaque, cyno) and/or human PSMA, comprising a heavy chain complementarity determining region 1 (HCDR1), a HCDR2 and a HCDR3 of SEQ ID NOs: 31, 44 and 45, respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs: 31, 44, and 45, respectively, and a light chain complementarity determining region 1 (LCDR1), a LCDR2 and a LCDR3 of SEQ ID NOs: 46, 29 and 27, respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis (cynomolgus monkey, macaque, cyno) and/or human PSMA, comprising a heavy chain complementarity determining region 1 (HCDR1), a HCDR2 and a HCDR3 of SEQ ID NOs: 36, 37 and 48, respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs: 36, 37, and 48, respectively, and a light chain complementarity determining region 1 (LCDR1), a LCDR2 and a LCDR3 of SEQ ID NOs: 49, 50 and 51, respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis (cynomolgus monkey, macaque, cyno) and/or human PSMA, comprising a heavy chain complementarity determining region 1 (HCDR1), a HCDR2 and a HCDR3 of SEQ ID NOs: 36, 37 and 52, respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs: 36, 37, and 52, respectively, and a light chain complementarity determining region 1 (LCDR1), a LCDR2 and a LCDR3 of SEQ ID NOs: 49, 50 and 51, respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis (cynomolgus monkey, macaque, cyno) and/or human PSMA, comprising a heavy chain complementarity determining region 1 (HCDR1), a HCDR2 and a HCDR3 of SEQ ID NOs: 8, 9 and 10, respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs: 8, 9, and 10, respectively, and a light chain complementarity determining region 1 (LCDR1), a LCDR2 and a LCDR3 of SEQ ID NOs: 11, 12 and 13, respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis (cynomolgus monkey, macaque, cyno) and/or human PSMA, comprising a heavy chain complementarity determining region 1 (HCDR1), a HCDR2 and a HCDR3 of SEQ ID NOs: 31, 32 and 33, respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs: 31, 32, and 33, respectively, and a light chain complementarity determining region 1 (LCDR1), a LCDR2 and a LCDR3 of SEQ ID NOs: 34, 12 and 35, respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis (cynomolgus monkey, macaque, cyno) and/or human PSMA, comprising a heavy chain complementarity determining region 1 (HCDR1), a HCDR2 and a HCDR3 of SEQ ID NOs: 53, 54 and 55, respectively.

The invention provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs: 53, 54, and 55, respectively, and a light chain complementarity determining region 1 (LCDR1), a LCDR2 and a LCDR3 of SEQ ID NOs: 23, 12 and 35, respectively.

The invention also provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA, comprising certain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences as described herein.

The invention also provides an isolated antibody and fragments thereof, specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA, comprising certain variable heavy chain region (VH) and variable light chain region (VL) amino acid sequences as described herein.

The invention also provides an isolated bispecific PSMA/CD3 antibody specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3.

The invention also provides an isolated bispecific PSMA/CD3 antibody specifically binding Pan troglodytes, Macaca fascicularis and/or human PSMA and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, comprising certain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, VH, VL, heavy chain or light chain amino acids sequences as described herein.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 42, 43, 11, 12 and 13, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 38, 39, 40 and 41, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 20, 21, 22, 23, 12 and 24, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 14, 15, 16, 17, 18 and 19, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 122, 123, 124, 23, 12, and 24, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 44, 45, 46, 29, and 47, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 48, 49, 50, and 51, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 52, 49, 50, and 51, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 8, 9, 10, 11, 12, and 13, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 32, 33, 34, 12, and 35, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 53, 54, 55, 23, 12, and 35, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 29, and 30, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 131, 29, and 132, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 133, and 132, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29, and 30, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR11, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29, and 136, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR11, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29, and 30, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 131, 29, and 132, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 133, and 132, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29, and 136, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 131, 29, and 132, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR11, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 133, and 132, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR11, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 137, 27, 28, 133, and 132, respectively.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises a heavy chain variable region (VH) of SEQ ID NO: 74 and a light chain variable region (VL) of SEQ ID NO: 61, and the second domain comprises the VH of SEQ ID NO: 104 and the VL of SEQ ID NO: 105.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises a heavy chain variable region (VH) of SEQ ID NO: 74 and a light chain variable region (VL) of SEQ ID NO: 61, and the second domain comprises the VH of SEQ ID NO: 152 and the VL of SEQ ID NO: 153.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises a heavy chain variable region (VH) of SEQ ID NO: 66 and a light chain variable region (VL) of SEQ ID NO: 67, and the second domain comprises the VH of SEQ ID NO: 104 and the VL of SEQ ID NO: 105.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises a heavy chain variable region (VH) of SEQ ID NO: 66 and a light chain variable region (VL) of SEQ ID NO: 67, and the second domain comprises the VH of SEQ ID NO: 152 and the VL of SEQ ID NO: 153.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises a heavy chain variable region (VH) of SEQ ID NO: 64 and a light chain variable region (VL) of SEQ ID NO: 65, and the second domain comprises the VH of SEQ ID NO: 104 and the VL of SEQ ID NO: 105.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises a heavy chain variable region (VH) of SEQ ID NO: 64 and a light chain variable region (VL) of SEQ ID NO: 65, and the second domain comprises the VH of SEQ ID NO: 152 and the VL of SEQ ID NO: 153.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises a heavy chain variable region (VH) of SEQ ID NO: 62 and a light chain variable region (VL) of SEQ ID NO: 63, and the second domain comprises the VH of SEQ ID NO: 104 and the VL of SEQ ID NO: 105.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises a heavy chain variable region (VH) of SEQ ID NO: 62 and a light chain variable region (VL) of SEQ ID NO: 63, and the second domain comprises the VH of SEQ ID NO: 152 and the VL of SEQ ID NO: 153.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises a heavy chain variable region (VH) of SEQ ID NO: 75 and a light chain variable region (VL) of SEQ ID NO: 76, and the second domain comprises the VH of SEQ ID NO: 104 and the VL of SEQ ID NO: 105.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises a heavy chain variable region (VH) of SEQ ID NO: 75 and a light chain variable region (VL) of SEQ ID NO: 76, and the second domain comprises the VH of SEQ ID NO: 152 and the VL of SEQ ID NO: 153.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises a heavy chain variable region (VH) of SEQ ID NO: 160 and a light chain variable region (VL) of SEQ ID NO: 65, and the second domain comprises the VH of SEQ ID NO: 104 and the VL of SEQ ID NO: 105.

The invention also provides an isolated bispecific PSMA/CD3 antibody and fragments thereof, comprising a first domain specifically binding PSMA and a second domain specifically binding CD3, wherein the first domain comprises a heavy chain variable region (VH) of SEQ ID NO: 160 and a light chain variable region (VL) of SEQ ID NO: 65, and the second domain comprises the VH of SEQ ID NO: 152 and the VL of SEQ ID NO: 153.

The invention also provides an immunoconjugate comprising the antibody or antigen-binding portion thereof of the invention linked to a therapeutic agent or to an imaging agent.

The invention also provides a pharmaceutical composition comprising the antibody of the invention and a pharmaceutically accepted carrier.

The invention also provides a polynucleotide encoding the antibody VH, the antibody VL or the antibody VH and the antibody VL of the invention.

The invention also provides a vector comprising the polynucleotide encoding the antibody VH, the antibody VL or the antibody VH and the VL of the invention.

The invention also provides a host cell comprising the vector of the invention.

The invention also provides a method of producing the antibody of the invention, comprising culturing the host cell of the invention in conditions that the antibody is expressed, and recovering the antibody produced by the host cell.

The invention also provides a method of treating a PSMA-overexpressing disease and/or cancer in a subject, comprising administering a therapeutically effective amount of the isolated antibody of the invention to the subject in need thereof for a time sufficient to treat the cancer.

The invention also provides a kit comprising the antibody of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show mammalian Fab supernatant titration curves for Anti-PSMA phage panning hits binding to LNCaP cells.

FIGS. 2A-2D show mammalian Fab supernatant titration curves for Anti-PSMA phage panning hits binding to Chimpanzee-PSMA expressing HEK cells.

FIGS. 3A-3D shows mammalian Fab supernatant titration curves for Anti-PSMA phage panning hits binding to Cynomolgus monkey PSMA-expressing HEK cells.

FIG. 4 shows the amino acid sequence of SP34 with sequential numbering. CDRs in AbM definition (K. R. Abhinandan and A. C. Martin, 2008. Mol. Immunol. 45, 3832-3839) are underlined. Ser230 is the last HC residue present in papain-cleaved Fab. Residues 231-455 are from IGHG3_MOUSE (mouse IgG3, isoform 2).

FIG. 5 shows the Human Framework Adaptation (“HFA”) variants for VH (SEQ ID NOS: 104, 102, 115, and 116, respectively, in order of appearance) and VL (SEQ ID NOS: 103, 117, and 105, respectively, in order of appearance). The numbering is sequential; CDRs in the AbM definition are underlined; residues that differ from SP34 are highlighted in bold; back mutations in HFA variants are bold and underlined. Figure discloses the sp34 VH and VL sequences as SEQ ID NOS 128 and 129, respectively.

FIG. 6 shows binding of SP34 HFA variants to primary Human T cells.

FIG. 7 shows binding of SP34 HFA variants to Cynomolgus primary T cells.

FIG. 8 shows that SP34 HFA variants activate primary human T cells in vitro. Negative controls are shown in white and positive controls are shown in black.

FIG. 9 shows that SP34 HFA variants activate primary cynomolgus T cells in vitro. Negative controls are shown in white and positive controls are shown in black. Non-CD3e-cross-reactive antibody G11 served as an additional negative control.

FIGS. 10A and 10B show the correlation of binding and activation by SP34 HFA variants. Average binding and CD69 Mean Fluorescence Intensity (“MFI”) values for human (FIG. 10A) and cynomolgus (FIG. 10B) were plotted against each other.

FIGS. 11A-11F show titration curves for PSMA×CD3 bispecific antibodies binding to LNCaP cells.

FIG. 12 shows titration curves for PSMA×CD3 bispecific antibodies binding to Chimpanzee PSMA-expressing HEK cells.

FIG. 13 shows titration curves for PSMA×CD3 bispecific antibodies binding to Cynomolgus monkey PSMA-expressing HEK cells.

FIG. 14 shows titration curves for PSMA×CD3 bispecific antibodies binding to human PSMA-expressing HEK cells.

FIG. 15A and FIG. 15B show titration curves for PSMA×CD3 bispecific antibodies for human PSMA-expressing HEK cells in a T-cell-mediated chromium release toxicity assay.

FIG. 16A and FIG. 16B show the comparison of medium vs high affinity CD3 arms in PSMA×CD3 bispecific antibodies in a T-cell-mediated chromium release toxicity assay for human PSMA-expressing HEK cells.

FIG. 17 shows titration curves for PSMA×CD3 bispecific antibodies for LNCaP cells in a T-cell-mediated chromium release toxicity assay.

FIG. 18 shows titration curves for PSMA×CD3 bispecific antibodies for Cynomolgus monkey PSMA-expressing HEK cells in a T-cell-mediated chromium release toxicity assay.

FIG. 19A shows titration curves for PS3B27 and control bispecific antibodies for human PSMA-expressing HEK cells in a T-cell-mediated Caspase 3/7 toxicity assay. FIG. 19B shows titration curves for PS3B27 and control bispecific antibodies for Cynomolgus monkey PSMA-expressing HEK cells in a T-cell-mediated Caspase 3/7 toxicity assay. FIG. 19C show titration curves for PS3B27 and control bispecific antibodies for LNCaP cells in a T-cell-mediated Caspase 3/7 toxicity assay.

FIG. 20 shows T-cell activation by PS3B27.

FIG. 21 shows prevention of tumorigenesis of HEK293-PSMA xenografts treated with PS3B27 or control bispecific antibodies in PBMC humanized NSG mice.

FIG. 22 shows Mean body weights of PBMC-humanized NSG mice bearing HEK293-PSMA xenografts with PS3B27 and control bispecific antibody treatment.

FIG. 23 shows efficacy of PS3B27 and control bispecific antibodies in tumorigenesis prevention of admixture HEK293-PSMA/T cell xenografts in male CD1 nude mice.

FIG. 24 shows Body Weight of CD1 male nude mice bearing Admixture HEK293-PSMA/T cell Xenografts Treated with PS3B27 and control bispecific antibodies.

FIG. 25 shows the overall structure of PSMB83 (AKA “PSMM84”) Fab bound to human PSMA ECD homodimer.

FIG. 26 shows a close view of PSMA main interactions with the PSMB83 (AKA “PSMM84”) Light Chain.

FIG. 27 shows a close view of PSMA main interactions with the PSMB83 (AKA “PSMM84”) Heavy Chain.

FIG. 28 shows the comparison of epitope residues of PSMB83 (AKA “PSMM84”) within the sequences of human (SEQ ID NO: 3), mouse (SEQ ID NO: 157) and Cynomolgus monkey (cyno) (SEQ ID NO: 2) PSMA. Epitope residues are shaded and sequence divergence is shown by underline.

FIG. 29 shows the paratope residues of PSMB83 (AKA “PSMM84”). CDRs are underlined and paratope residues are shaded. Figure discloses SEQ ID NOS 158 and 159, respectively, in order of appearance.

FIG. 30 shows an interaction map with direct contacts made between PSMA and PSMB83 (AKA “PSMM84”). Van der Waals interactions are shown as dashed lines and H-bonds are solid lines with arrows pointing to the backbone atoms.

FIG. 31 shows expression levels of anti-PSMA Fab clones derived from PSMB83 as compared to expression of parent PSMB83. Raw luminescence numbers were plotted against the log concentration.

FIG. 32 shows binding to human PSMA of anti-PSMA Fab clones derived from PSMB83 as compared to binding of parent PSMB83. Raw luminescence numbers were plotted against the log concentration.

FIG. 33 shows binding to cyno PSMA of anti-PSMA Fab clones derived from PSMB83 as compared to binding of parent PSMB83. Raw luminescence numbers were plotted against the log concentration.

FIG. 34 shows binding to human PSMA of anti-PSMA Fab clones derived from PSMB83 as compared to binding of parent PSMB83. Raw luminescence numbers were normalized by Fab expression levels.

FIG. 35 shows binding to cyno PSMA of anti-PSMA Fab clones derived from PSMB83 as compared to binding of parent PSMB83. Raw luminescence numbers were normalized by Fab expression levels.

FIG. 36 shows LNCAP cell binding of a subset of affinity-matured PSMA×CD3 bispecific antibodies.

FIG. 37 shows LNCAP cell binding of a subset of affinity-matured PSMA×CD3 bispecific antibodies.

FIG. 38 shows PSMA-negative PC3 cell binding results of affinity-matured PSMA×CD3 bispecific antibodies.

FIG. 39 shows results of PSMA×CD3 Affinity Matured Bispecific Abs in a Functional Cell Killing Assay.

FIG. 40 shows anti-tumor efficacy of PS3B79 in LnCAP AR.TB human prostate xenografts in T cell humanized NSG mice. Subcutaneous LnCAP AR.TB tumors were measured twice weekly and the results presented as the mean tumor volume, expressed in mm³±SEM (*, p<0.0001).

FIG. 41 shows anti-tumor efficacy of PS3B90 in LnCAP AR.TB human prostate xenografts in T cell humanized NSG mice. Subcutaneous LnCAP AR.TB tumors were measured twice weekly and the results presented as the mean tumor volume, expressed in mm³±SEM (*, p<0.001).

FIG. 42 shows the effect of PS3B72 (PSMA×CD3) on Established Patient-Derived Xenograft LuCaP 86.2 Prostate Tumor Model in T-Cell Humanized NSG Mice. Subcutaneous LuCaP 86.2 tumors were measured twice weekly and the results presented as the mean tumor volume, expressed in mm³±SEM (*, p<0.0001).

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

“Specific binding” or “specifically binds” or “binds” refers to an antibody binding to an antigen or an epitope within the antigen with greater affinity than for other antigens. Typically, the antibody binds to the antigen or the epitope within the antigen with an equilibrium dissociation constant (K_(D)) of about 5×10⁻⁸ M or less, for example about 1×10⁻⁹ M or less, about 1×10⁻¹⁰ M or less, about 1×10⁻¹¹ M or less, or about 1×10⁻² M or less, typically with the K_(D) that is at least one hundred fold less than its K_(D) for binding to a non-specific antigen (e.g., BSA, casein). The dissociation constant may be measured using standard procedures. Antibodies that specifically bind to the antigen or the epitope within the antigen may, however, have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno) or Pan troglodytes (chimpanzee, chimp). While a monospecific antibody specifically binds one antigen or one epitope, a bispecific antibody specifically binds two distinct antigens or two distinct epitopes.

“Antibodies” is meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity. “Full length antibody molecules” are comprised of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds as well as multimers thereof (e.g. IgM). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CH1, hinge, CH2 and CH3). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The VH and the VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-to-carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

“Complementarity determining regions (CDR)” are “antigen binding sites” in an antibody. CDRs may be defined using various terms: (i) Complementarity Determining Regions (CDRs), three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3) are based on sequence variability (Wu and Kabat, (1970) J Exp Med 132:211-50; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) “Hypervariable regions”, “HVR”, or “HV”, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3) refer to the regions of an antibody variable domains which are hypervariable in structure as defined by Chothia and Lesk (Chothia and Lesk, (1987) Mol Biol 196:901-17). The International ImMunoGeneTics (IMGT) database (http://www_imgt_org) provides a standardized numbering and definition of antigen-binding sites. The correspondence between CDRs, HVs and IMGT delineations is described in Lefranc et al., (2003) Dev Comparat Immunol 27:55-77. The term “CDR”, “HCDR1”, “HCDR2”, “HCDR3”, “LCDR1”, “LCDR2” and “LCDR3” as used herein includes CDRs defined by any of the methods described supra, Kabat, Chothia or IMGT, unless otherwise explicitly stated in the specification.

Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate species may assigned to one of two clearly distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

“Antibody fragments” or “antigen-binding fragments” refer to a portion of an immunoglobulin molecule that retains the heavy chain and/or the light chain antigen binding site, such as heavy chain complementarity determining regions (HCDR) 1, 2 and 3, light chain complementarity determining regions (LCDR) 1, 2 and 3, a heavy chain variable region (VH), or a light chain variable region (VL). Antibody or antigen-binding fragments include well known Fab, F(ab′)2, Fd and Fv fragments as well as domain antibodies (dAb) consisting of one VH domain. VH and VL domains may be linked together via a synthetic linker to form various types of single chain antibody designs where the VH/VL domains may pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in Int. Patent Publ. Nos. WO1998/44001, WO1988/01649, WO1994/13804 and WO1992/01047.

“Monoclonal antibody” refers to an antibody population with single amino acid composition in each heavy and each light chain, except for possible well known alterations such as removal of C-terminal lysine from the antibody heavy chain. Monoclonal antibodies typically bind one antigenic epitope, except that bispecific monoclonal antibodies bind two distinct antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibody may be monospecific or multispecific, or monovalent, bivalent or multivalent. A bispecific antibody is included in the term monoclonal antibody.

“Isolated antibody” refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody specifically binding PSMA is substantially free of antibodies that specifically bind antigens other than PSMA). In the case of bispecific PSMA×CD3 antibodies, the bispecific antibody specifically binds both PSMA and CD3, and is substantially free of antibodies that specifically bind antigens other that PSMA and CD3. “Isolated antibody” encompasses antibodies that are isolated to a higher purity, such as antibodies that are 800/%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 960%, 97%, 98%, 99% or 100% pure.

“Humanized antibody” refers to an antibody in which the antigen binding sites are derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Humanized antibody may include substitutions in the framework so that the framework may not be an exact copy of expressed human immunoglobulin or human immunoglobulin germline gene sequences.

“Human antibody” refers to an antibody having heavy and light chain variable regions in which both the framework and the antigen binding site are derived from sequences of human origin. If the antibody contains a constant region or a portion of the constant region, the constant region also is derived from sequences of human origin.

Human antibody comprises heavy or light chain variable regions that are “derived from” sequences of human origin if the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such exemplary systems are human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice or rats carrying human immunoglobulin loci as described herein. “Human antibody” may contain amino acid differences when compared to the human germline immunoglobulin or rearranged immunoglobulin genes due to for example naturally occurring somatic mutations or intentional introduction of substitutions into the framework or antigen binding site, or both. Typically, “human antibody” is at least about 80%, 81%, 82%/0, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical in amino acid sequence to an amino acid sequence encoded by human germline immunoglobulin or rearranged immunoglobulin genes. In some cases, “human antibody” may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knappik et al., (2000) J Mol Biol 296:57-86, or synthetic HCDR3 incorporated into human immunoglobulin gene libraries displayed on phage, for example as described in Shi et al., (2010) J Mol Biol 397:385-96, and in Int. Patent Publ. No. WO2009/085462.

Human antibodies derived from human immunoglobulin sequences may be generated using systems such as phage display incorporating synthetic CDRs and/or synthetic frameworks, or may be subjected to in vitro mutagenesis to improve antibody properties, resulting in antibodies that are not expressed by the human antibody germline repertoire in vivo.

Antibodies in which antigen binding sites are derived from a non-human species are not included in the definition of“human antibody”.

“Recombinant” refers to DNA, antibodies and other proteins that are prepared, expressed, created or isolated by recombinant means when segments from different sources are joined to produce recombinant DNA, antibodies or proteins.

“Epitope” refers to a portion of an antigen to which an antibody specifically binds. Epitopes typically consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be composed of contiguous and/or discontiguous amino acids that form a conformational spatial unit. For a discontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3-dimensional space through the folding of the protein molecule. Antibody “epitope” depends on the methodology used to identify the epitope.

“Paratope” refers to a portion of an antibody to which an antigen specifically binds. A paratope may be linear in nature or may be discontinuous, formed by a spatial relationship between non-contiguous amino acids of an antibody rather than a linear series of amino acids. A “light chain paratope” and a “heavy chain paratope” or “light chain paratope amino acid residues” and “heavy chain paratope amino acid residues” refer to antibody light chain and heavy chain residues in contact with an antigen, respectively, or in general, “antibody paratope residues” refer to those antibody amino acids that are in contact with antigen.

“Bispecific” refers to an antibody that specifically binds two distinct antigens or two distinct epitopes within the same antigen. The bispecific antibody may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between two or more distinct antigens.

“Multispecific” refers to an antibody that specifically binds two or more distinct antigens or two or more distinct epitopes within the same antigen. The multispecific antibody may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between two or more distinct antigens.

“Variant” refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications, for example one or more substitutions, insertions or deletions.

“Vector” refers to a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers, that function to facilitate the duplication or maintenance of these polynucleotides in a biological system, such as a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The vector polynucleotide may be DNA or RNA molecules or a hybrid of these, single stranded or double stranded.

“Expression vector” refers to a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

“Polynucleotide” refers to a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. Double and single-stranded DNA and RNA are typical examples of polynucleotides. “Polypeptide” or “protein” refers to a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than 50 amino acids may be referred to as “peptides”.

“Flow cytometry” is a technology that is used to analyze the physical and chemical characteristics of particles in a fluid as it passes through at least one laser. Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths (Adan, et al, Critical Reviews in Biotechnology (2016) 1549-7801).

“Anti-idiotypic (anti-Id) antibody” is an antibody which recognizes the antigenic determinants (e.g. the paratope or CDRs) of the antibody. It is generally known in the art the process of producing or preparing an anti-idiotypic antibody. (Lathey, J. et al Immunology 1986 57(1):29-35). The anti-Id antibody may be antigen-blocking or non-blocking. The antigen-blocking anti-Id antibody may be used to detect the free antibody in a sample (e.g. anti-PSMA, anti-CD3 or the bispecific PSMA×CD3 antibody of the invention described herein). The non-blocking anti-Id antibody may be used to detect the total antibody (free, partially bound to antigen, or fully bound to antigen) in a sample. An anti-Id antibody may be prepared by immunizing an animal with the antibody to which an anti-Id antibody is being prepared. In some embodiments described herein, the anti-idiotypic antibody is used for detecting the level of the therapeutic antibodies (e.g. anti-PSMA, anti-CD3 or the bispecific PSMA×CD3 antibody of the invention described herein) in a sample.

An anti-Id antibody may also be used as an immunogen to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. An anti-anti-Id may be epitopically identical to the original mAb, which induced the anti-Id antibody. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to identify other clones expressing antibodies of identical specificity. Anti-Id antibodies may be varied (thereby producing anti-Id antibody variants) and/or derivatized by any suitable technique, such as those described elsewhere herein with respect to the antibodies specifically binding PSMA or CD3, or the bispecific PSMA×CD3 antibodies.

PSMA refers to Prostate Specific Membrane Antigen. The amino acid sequence of the Pan troglodytes (also refered to as chimpanzee or chimp) PSMA is shown in SEQ ID NO: 1. The extracellular domain spans residues 44-750, the transmembrane domain spans residues 20-43 and the cytoplasmic domain spans residues 1-19 of SEQ ID NO: 1. The amino acid sequence of the Macaca fascicularis (also refered to as cynomolgus monkey, macaque or cyno) PSMA is shown in SEQ ID NO: 2. The extracellular domain spans residues 44-750, the transmembrane domain spans residues 20-43 and the cytoplasmic domain spans residues 1-19 of SEQ ID NO: 2. The amino acid sequence of the human PSMA is shown in SEQ ID NO: 3. The extracellular domain spans residues 44-750, the transmembrane domain spans residues 20-43 and the cytoplasmic domain spans residues 1-19 of SEQ ID NO: 3.

CD3 refers to the T-cell antigen receptor. Throughout the specification, “CD3-specific” refers to antibodies that bind specifically to the T-cell receptor complex. More specifically, the antibodies bind to the CD3-epsilon polypeptide, which together with CD3-gamma, -delta and -zeta, and the T-cell receptor alpha/beta and gamma/delta heterodimers, forms the T-cell receptor-CD3 complex. This complex plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. The CD3 complex mediates signal transduction, resulting in T cell activation and proliferation. CD3 is required for the immune response.

“In combination with” means that two or more therapeutics are administered to a subject together in a mixture, concurrently as single agents or sequentially as single agents in any order.

“Overexpress”, “overexpressed” and “overexpressing” interchangeably refers to a sample such as a cancer cell, malignant cell or cancer tissue that has measurably higher levels of PSMA when compared to a reference sample. The overexpression may be caused by gene amplification or by increased transcription or translation. Expression and overexpression of protein in the sample may be measured using well known assays using, for example ELISA, immunofluorescence, flow cytometry or radioimmunoassay on live or lysed cells. Expression and overexpression of a polynucleotide in the sample may be measured, for example, using fluorescent in situ hybridization, Southern blotting, or PCR techniques. A protein or a polynucleotide is overexpressed when the level of the protein or the polynucleotide in the sample is at least 1.5-fold higher when compared to the reference sample. Selection of the reference sample is well known.

“Sample” refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples are of biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tear drops, feces, sputum, mucosal secretions of the secretory tissues and organs, vaginal secretions, ascites fluids such as those associated with non-solid tumors, fluids of the pleural, pericardial, peritoneal, abdominal and other body cavities, fluids collected by bronchial lavage, liquid solutions contacted with a subject or biological source, for example, cell and organ culture medium including cell or organ conditioned medium, lavage fluids and the like, tissue biopsies, fine needle aspirations or surgically resected tumor tissue.

A “cancer cell” or a “tumor cell” as used herein refers to a cancerous, pre-cancerous or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes. These changes do not necessarily involve the uptake of new genetic material. Although transformation may arise from infection with a transforming virus and incorporation of new genomic nucleic acid or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation/cancer is exemplified by morphological changes, immortalization of cells, aberrant growth control, foci formation, proliferation, malignancy, modulation of tumor specific marker levels, invasiveness, tumor growth in suitable animal hosts such as nude mice, and the like, in vitro, in vivo, and ex vivo (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed. 1994)). Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ±10% of the recited value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

“Effector antigens” are antigens from cells of the immune system which can stimulate or trigger cytotoxicity, phagocytosis, antigen presentation, cytokine release. Such effector antigens are from, for example but not limited to, T cells and natural killer (NK) cells. Examples of suitable specificities for effector antigens include but are not limited to CD3 or CD3 subunits such as CD3ε for T cells and CD16 for NK cells. Such cell surface molecules of effector cells are suitable for mediating cell killing. Effector cells are cells of the immune system which can stimulate or trigger cytotoxicity, phagocytosis, antigen presentation, cytokine release. Such effector cells are, for example but not limited to, T-cells, natural killer (NK) cells, granulocytes, monocytes, macrophages, dendritic cells, and antigen-presenting cells. Examples of suitable specificities for effector cells include but are not limited to CD2, CD3 and CD3 subunits such as CD3e, CD5, CD28 and other components of the T-cell receptor (TCR) for T cells; CD16, CD16A, CD25, CD38, CD44, CD56, CD69, CD94, CD335 (NKp46), CD336, (NKp44), CD337 (NKp30), NKp80, NKG2C and NKG2D, DNAM, NCRs for NK cells; CD18, CD64 and CD89 for granulocytes; CD18, CD32, CD64, CD89 and mannose receptor for monocytes and macrophages; CD64 and mannose receptor for dendritic cells; as well as CD35. In certain embodiments of the inventions, those specificities, i. e. cell surface molecules, of effector cells are suitable for mediating cell killing upon binding of a bispecific or multispecific molecules to such cell surface molecule and, thereby, inducing cytolysis or apoptosis.

“Bispecific PSMA×CD3 antibody”, “PSMA/CD3 antibody”, “bispecific anti-PSMA×CD3 antibody” or “anti-PSMA/CD3 antibody” refers to a molecule comprising at least one binding domain specifically binding PSMA and at least one binding domain specifically binding CD3. The domains specifically binding PSMA and CD3 are typically VH/VL pairs. The bispecific anti-PSMA×CD3 antibody may be monovalent in terms of its binding to either PSMA or CD3.

“Valent” refers to the presence of a specified number of binding sites specific for an antigen in a molecule. As such, the terms “monovalent”, “bivalent”, “tetravalent”, and “hexavalent” refer to the presence of one, two, four and six binding sites, respectively, specific for an antigen in a molecule. “Multivalent” refers to the presence of two or more binding sites specific for an antigen in a molecule.

“An antigen specific CD4+ or CD8+ T cell” refers to a CD4+ or CD8+ T cell activated by a specific antigen, or immunostimulatory epitope thereof.

“Subject” includes any human or nonhuman animal. “Nonhuman animal” includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows chickens, amphibians, reptiles, etc. Except when noted, the terms “patient” or “subject” are used interchangeably.

The numbering of amino acid residues in the antibody constant region throughout the specification is according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), unless otherwise explicitly stated.

Conventional one and three-letter amino acid codes are used herein as shown in Table 1.

TABLE 1 Amino acid Three-letter code One-letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys C Glutamate Gln E Glutamine Glu Q Glycine Gly G Histidine His H Isoleucine Ile I Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

Compositions of Matter

The present invention provides antibodies and fragments thereof that specifically bind PSMA and multispecific antibodies that specifically bind PSMA and CD3 and fragments thereof. The present invention provides polypeptides and polynucleotides encoding the antibodies of the invention or complementary nucleic acids thereof, vectors, host cells, and methods of making and using them.

The antibodies and fragments thereof that bind to PSMA bind to the chimpanzee target antigen. In one embodiment, the antibodies and fragments thereof bind to the human and macaque PSMA target antigens with affinities within 5-fold of each other. In other words, the difference in antibody binding is less than a multiple of 5. In this case, the identical antibody molecule can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of PSMA in primates and as a drug in humans. Put in other words, the same PSMA-specific molecule can be used in preclinical animal studies as well as in clinical studies in humans. This leads to highly comparable results and a much-increased predictive power of the animal studies compared to species-specific surrogate molecules. Since the PSMA domain is cross-species specific, i.e. reactive with the human and macaque antigens, the antibody or fragments thereof of the invention can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and—in the identical form—as drug in humans.

The present invention also provides for multispecific antibodies that specifically bind to PSMA. According to the invention, a bispecific, i. e. bifunctional, antibody can be used to engage two different therapeutic targets or perform two distinct functions. Such antibodies can be used for example to recruit an immune effector cell, e.g. T- or NK-cell, towards a particular target cell. Various antibody-fragment based molecules are known and under investigation, for example for cancer therapy. A multispecific antibody of the invention may be a trispecific antibody for dual targeting of tumor cells—these are trifunctional structures that can be designed to target two different targets/epitopes on the tumor cell and with the third functionality bind with high affinity to either T-cells or NK-cells. Trispecific antibodies targeting two distinct tumor epitopes and engaging T- or NK-cells lyse the tumor cells that express both targets. Such molecules can be generated by antibody formats known in the art and are fully described. (WO20151842071, WO2015158636, WO2010136172, WO2013174873). In a trispecific antibody embodiment of the invention, the multispecific antibody may be specific for PSMA and a second distinct antigen on the same or another tumor cell and additionally specific for an effector cell, in particular a T cell or an NK cell.

The present invention also provides for a PSMA X “effector antigen” bispecific antibody. In one embodiment, the effector antigen of the the PSMA X “effector antigen” bispecific antibody is CD3. It has been found in the present invention that it is possible to generate a PSMA×CD3 bispecific antibody wherein the identical molecule can be used in preclinical animal testing, as well as clinical studies and even in therapy in human. This is due to the identification of the PSMA×CD3 bispecific antibody, which, in addition to binding to human PSMA and human CD3, respectively, also binds to the homologs of antigens of chimpanzee and macaques. The PSMA×CD3 bispecific antibody of the invention can be used as a therapeutic agent against various diseases, including, but not limited, to cancer. In view of the above, the need to construct a surrogate target PSMA×CD3 bispecific antibody for testing in a phylogenetically distant (from humans) species disappears. As a result, the identical molecule can be used in animal preclinical testing as is intended to be administered to humans in clinical testing as well as following market approval and therapeutic drug administration. The ability to use the same molecule for preclinical animal testing as in later administration to humans virtually eliminates, or at least greatly reduces, the danger that the data obtained in preclinical animal testing have limited applicability to the human case. In short, obtaining preclinical safety data in animals using the same molecule as will actually be administered to humans does much to ensure the applicability of the data to a human-relevant scenario. In contrast, in conventional approaches using surrogate molecules, said surrogate molecules have to be molecularly adapted to the animal test system used for preclinical safety assessment. Thus, the molecule to be used in human therapy in fact differs in sequence and also likely in structure from the surrogate molecule used in preclinical testing in pharmacokinetic parameters and/or biological activity, with the consequence that data obtained in preclinical animal testing have limited applicability/transferability to the human case. The use of surrogate molecules requires the construction, production, purification and characterization of a completely new construct. This leads to additional development costs and time necessary to obtain that molecule. In sum, surrogates have to be developed separately in addition to the actual drug to be used in human therapy, so that two lines of development for two molecules have to be carried out. Therefore, a major advantage of the PSMA×CD3 bispecific antibody of the invention exhibiting cross-species specificity described herein is that the identical molecule can be used for therapeutic agents in humans and in preclinical animal testing.

Another major advantage of the antibody and multispecific antibody of the invention is its applicability for preclinical testing in various primates. The behavior of a drug candidate in animals should ideally be indicative of the expected behavior of this drug candidate upon administration to humans. As a result, the data obtained from such preclinical testing should therefore generally have a highly predictive power for the human case. However, as learned from the tragic outcome of the recent Phase I clinical trial on TGN1412 (a CD28 monoclonal antibody), a drug candidate may act differently in a primate species than in humans: Whereas in preclinical testing of said antibody, no or only limited adverse effects have been observed in animal studies performed with cynomolgus monkeys, six human patients developed multiple organ failure upon administration of said antibody (Lancet 368 (2006), 2206-7). The results of these dramatic, non-desired negative events suggest that it may not be sufficient to limit preclinical testing to only one (non-chimpanzee primate) species. The fact that the described antibody and multispecific antibody specifically bind PSMA of chimpanzee and cynomolgus monkey may help to overcome the problems faced in the case mentioned above. Accordingly, the present invention provides means and methods for minimizing species differences in effects when drugs for human therapy are being developed and tested.

With the antibody and multi specific antibody of the invention it is also no longer necessary to adapt the test animal to the drug candidate intended for administration to humans, such as e.g. the creation of transgenic animals. The cross-species specificity of the PSMA antibody or multispecific antibody of the invention allows the antibody to be directly used for preclinical testing in non-chimpanzee primates without any genetic manipulation of the animals. As well known to those skilled in the art, approaches in which the test animal is adapted to the drug candidate always bear the risk that the results obtained in the preclinical safety testing are less representative and predictive for humans due to the modification of the animal. For example, in transgenic animals, the proteins encoded by the transgenes are often highly over-expressed. Thus, data obtained for the biological activity of an antibody against this protein antigen may be limited in their predictive value for humans in which the protein is expressed at much lower, more physiological levels.

A further advantage of the uses of the antibody of the invention exhibiting cross-species specificity is the fact that the use of chimpanzees, an endangered species, can be avoided for animal testing. Chimpanzees are the closest relatives to humans and were recently grouped into the family of hominids based on the genome sequencing data (Wildman et al., PNAS 100 (2003), 7181). Therefore, data obtained with chimpanzee is generally considered to be highly predictive for humans. However, due to their status as endangered species, the number of chimpanzees, which can be used for medical experiments, is highly restricted. As stated above, maintenance of chimpanzees for animal testing is therefore both costly and ethically problematic. The uses of the antibody of the invention avoid both ethical objections and financial burden during preclinical testing without prejudicing the quality, i.e. applicability, of the animal testing data obtained. In light of this, the uses of the antibody or multispecific antibody specifically binding PSMA of the invention provide for a reasonable alternative for studies in chimpanzees.

A still further advantage of the antibody or multispecific antibody specifically binding PSMA of the invention is the ability of extracting multiple blood samples when using it as part of animal preclinical testing, for example in the course of pharmacokinetic animal studies. Multiple blood extractions can be much more readily obtained with a non-chimpanzee primate than with lower animals, e.g. a mouse. The extraction of multiple blood samples allows continuous testing of blood parameters for the determination of the biological effects induced by the antibody or multispecific antibody specifically binding PSMA of the invention. Furthermore, the extraction of multiple blood samples enables the researcher to evaluate the pharmacokinetic profile of the antibody or multispecific antibody specifically binding PSMA of the invention as defined herein. In addition, potential side effects, which may be induced by said antibody or multispecific antibody specifically binding PSMA of the invention reflected in blood parameters can be measured in different blood samples extracted during the course of the administration of said antibody.

This allows the determination of the potential toxicity profile of antibody or multispecific antibody binding PSMA of the invention as defined herein.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the isolated antibody or antibody fragment thereof specifically binding PSMA has one, two, three, four or five of the following properties:

-   -   a) binds Pan troglodytes PSMA extracellular domain (ECD) with an         equilibrium dissociation constant (K_(D)) of 25 nM or less,         wherein the K_(D) is measured using ProteOn XPR36 system at +25°         C.,     -   b) binds LNCaP cells with a calculated EC₅₀ of 20 nM or less and         binds Macaca fascicularis PSMA-expressing HEK cells with a         calculated EC₅₀ of 40 nM or less, wherein the difference in         calculated EC₅₀ between binding LNCaP cells and binding Macaca         fascicularis PSMA-expressing HEK cells is less than 5-fold, and         wherein the calculated EC₅₀ is measured in a whole cell binding         assay at 0° C. using flow cytometry,     -   c) binds recombinant PSMA ECD from human (SEQ ID NO: 7), Pan         troglodytes (SEQ ID NO:4) and Macaca fascicularis (SEQ ID NO: 5)         with an equilibrium dissociation constant (K_(D)) of 12 nM or         less, wherein the K_(D) is measured using Proteon surface         plasmon resonance assay ProteOn XPR36 system at +25C;     -   d) displays T-cell mediated killing of LNCaP cells, C42 cells,         human PSMA-expressing HEK cells or Macaca fascicularis         PSMA-expressing HEK cells when paired in a bispecific antibody         with anti-CD3 antibody CD3B219, wherein the T-cell mediated         killing is measured by Chromium-51 or by caspase 3/7 activation         assay, or     -   e) recognizes a conformational epitope wherein the epitope is         comprised of residues 1138, F235, P237, G238, D244, Y299, Y300,         Q303, K304, E307, and K324-P326 of human PSMA (SEQ ID NO:3)

Exemplary such antibodies or fragments thereof are PSMA antibodies PSMB119, PSMB120, PSMB121, PSMB122, PSMB123, PSMB87, PSMB126, PSMB127, PSMB128, PSMB129, PSMB130, PSMB120, PSMB121, PSMB122, PSMB123, PSMB127, PSMB128, PSMB130, PSMB344, PSMB345, PSMB346, PSMB347, PSMB349, PSMB358, PSMB359, PSMB360, PSMB361, PSMB362, PSMB363, and PSMB365 described herein.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the isolated PSMA antibody or antibody fragment thereof specifically binding PSMA binds Chimpanzee PSMA ECD with an equilibrium dissociation constant (K_(D)) of about 30 nM or less, wherein the K_(D) is measured using ProteOn XPR36 system at +25° C. as described in Example 8. Assays for measuring affinity by SPR using Proteon include assays where the assay is performed at room temperature (e.g. at or near 25° C.), wherein the antibody capable of binding to chimp PSMA ECD is captured on the Proteon sensor chip by an anti-Fc antibody (e.g. (Jackson ImmunoResearch Laboratory, cat #109-005-098) to a level around 100RUs, followed by injection of recombinant PSMA ECD, and the collection of association and dissociation data at a flow rate of 50 μl/min.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the isolated PSMA antibody or antibody binding fragment thereof specifically binding PSMA binds LNCaP cells with an EC₅₀ of 20 nM or less and binds cyno PSMA expressing HEK cells with an EC₅₀ of 40 nM or less, wherein the difference in calculated EC₅₀ between binding LNCaP cells and binding cyno PSMA-expressing HEK cells is less than 5-fold, wherein the cell binding is measure using FACS as described in Example 7. Assays for measuring whole cell binding by FACS are performed at a density of 200,000 cells per well for 1 hour on ice. The amount of antibody bound to whole cells is detected with a labelled secondary antibody, for example, with a mouse anti-human kappa-RPE antibody (Life Technologies cat # MH10514) by a FACS Array flow cytometer.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the isolated antibody or antibody fragment thereof specifically binding PSMA binds human, chimp and cyno PSMA ECDs with an equilibrium dissociation constant (K_(D)) of 12 nM or less, wherein the K_(D) is measured using ProteOn XPR36 system at +25° C. as described in Example 8. Assays for measuring affinity by SPR using Proteon include assays where the assay is performed at room temperature (e.g. at or near 25° C.), wherein the antibody capable of binding to chimp PSMA ECD is captured on the Proteon sensor chip by an anti-Fc antibody (e.g. (Jackson ImmunoResearch Laboratory, cat #109-005-098) to a level around 100RUs, followed by injection of recombinant PSMA ECD, and the collection of association and dissociation data at a flow rate of 50 μl/min.

The measured affinity of a particular antibody/PSMA interaction may vary if measured under different conditions (e.g., osmolarity, pH). Thus, measurements of affinity and other binding parameters (e.g., K_(D), K_(on), K_(off)) are typically made with standardized conditions and a standardized buffer, such as the buffer described herein. Skilled in the art will appreciate that the internal error for affinity measurements for example using Biacore 3000 or ProteOn (measured as standard deviation, SD) may typically be within 5-33% for measurements within the typical limits of detection. Therefore the term “about” in the context of K_(D) reflects the typical standard deviation in the assay. For example, the typical SD for a K_(D) of 1×10⁻⁹ M is up to +0.33×10⁻⁹ M.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the isolated antibody or antibody fragment thereof specifically binding PSMA show T-cell mediated killing of human PSMA-expressing LNCaP cells, C42cells, human PSMA-expressing HEK cells or cyno PSMA-expressing HEK cells when paired in a bispecific antibody with anti-CD3 antibody CD3B219, wherein the T-cell mediated killing is measured by Chromium-51 release and target cells are cultured with pre-activated T-cells at 5:1 ratio for 18-24 hours or by caspase 3/7 activation assay as in Example 6. In some embodiments, the isolated antibody or antibody fragment thereof specifically binding PSMA show T-cell mediated killing of human PSMA-expressing LNCaP and C42 cells with an EC₅₀ of about 0.3-0.5 nM or less and 0.12-0.03 nM or less, respectively, when paired in a bispecific antibody with anti-CD3 antibody CD3B219, wherein the T-cell mediated killing is measured by caspase 3/7 activation assay as in Example 9. Target PSMA-expressing cells are cultured with pre-activated T-cells at 1:3 ratio for 18-24 hours and cleavage of the added Caspase 3/7 substrate results in a fluorescent DNA dye, with fluorescence restricted to the cell nucleus.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the isolated antibody or antibody fragment thereof specifically binding PSMA recognizes a conformational epitope wherein the epitope is comprised of residues 1138, F235, P237, G238, D244, Y299, Y300, Q303, K304, E307, and K324-P326 as determined by X-ray crystallography as described in Example 13.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody or fragment thereof specifically binding PSMA of the invention comprises the HCDR1, the HCDR2 and the HCDR3 contained within a heavy chain variable region (VH) of SEQ ID NOs: 60, 62, 64, 66, 68, 70, 72, 74, 75, 77, 79, 160, 138, 139, or 140, wherein the HCDR1, the HCDR2 and the HCDR3 are defined by Chothia, Kabat, or IMGT.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody or fragments thereof specifically binding PSMA of the invention comprises the LCDR1, the LCDR2 and the LCDR3 contained within a light chain variable region (VL) of SEQ ID NOs: 61, 63, 65, 67, 69, 71, 73, 76, 78, 142, 143, or 144, wherein the LCDR1, the LCDR2 and the LCDR are defined by Chothia, Kabat, or IMGT.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises

-   -   the HCDR1 of SEQ ID NOs: 8, 14, 20, 25, 31, 36, 46, 53, or 122;     -   the HCDR2 of SEQ ID NOs: 9, 15, 21, 26, 32, 37, 42, 44, 54, 123,         130, 134, 135, or 137; and     -   the HCDR3 of SEQ ID NOs: 10, 16, 22, 27, 33, 38, 43, 45, 48, 52,         55, 124.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises

-   -   the LCDR1 of SEQ ID NOs: 11, 17, 23, 28, 34, 39, 46, 49, or 131;     -   the LCDR2 of SEQ ID NOs: 12, 18, 29, 40, 50, or 133; and     -   the LCDR3 of SEQ ID NOs: 13, 19, 24, 30, 35, 41, 47, 51, 132, or         136.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises

-   -   the HCDR1 of SEQ ID NOs: 8, 14, 20, 25, 31, 36, 53, or 122;     -   the HCDR2 of SEQ ID NOs: 9, 15, 21, 26, 32, 37, 42, 44, 54, 123,         130, 134, 135, or 137;     -   the HCDR3 of SEQ ID NOs: 10, 16, 22, 27, 33, 38, 43, 45, 48 51,         52, 55, or 124;     -   the LCDR1 of SEQ ID NOs: 11, 17, 23, 28, 34, 39, 46, 49, or 131;     -   the LCDR2 of SEQ ID NOs: 12, 18, 29, 40, 50, or 133; and     -   the LCDR3 of SEQ ID NOs: 13, 19, 24, 30, 35, 41, 47, 51, 132, or         136.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2 and the HCDR3 of

-   -   SEQ ID NOs: 8, 9 and 10, respectively;     -   SEQ ID NOs: 14, 15 and 16, respectively;     -   SEQ ID NOs: 20, 21 and 22, respectively,     -   SEQ ID NOs: 25, 26 and 27, respectively;     -   SEQ ID NOs: 25, 130 and 27, respectively;     -   SEQ ID NOs: 25, 134 and 27, respectively;     -   SEQ ID NOs: 25, 135 and 27, respectively;     -   SEQ ID NOs: 25, 137 and 27, respectively;     -   SEQ ID NOs: 31, 32 and 33, respectively;     -   SEQ ID NOs: 36, 37 and 38, respectively;     -   SEQ ID NOs: 31, 42 and 43, respectively;     -   SEQ ID NOs: 31, 44 and 45, respectively,     -   SEQ ID NOs: 36, 37 and 48, respectively;     -   SEQ ID NOs: 36, 37 and 52, respectively;     -   SEQ ID NOs: 53, 54 and 55, respectively; or     -   SEQ ID NOs: 122, 123, and 124, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the LCDR1, the LCDR2 and the LCDR3 of

-   -   SEQ ID NOs: 11, 12 and 13, respectively;     -   SEQ ID NOs: 17, 18 and 19, respectively;     -   SEQ ID NOs: 23, 12 and 24, respectively;     -   SEQ ID NOs: 28, 29 and 30, respectively;     -   SEQ ID NOs: 28, 29 and 136, respectively;     -   SEQ ID NOs: 28, 133 and 132, respectively;     -   SEQ ID NOs: 34, 12 and 35, respectively;     -   SEQ ID NOs: 39, 40 and 41, respectively;     -   SEQ ID NOs: 46, 29 and 47, respectively;     -   SEQ ID NOs: 49, 50 and 51, respectively;     -   SEQ ID NOs: 23, 12 and 35, respectively; or     -   SEQ ID NOs: 131, 29 and 132, respectively;

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 8, 9, 10, 11, 12 and 13, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 14, 15, 16, 17, 18 and 19, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 20, 21, 22, 23, 12 and 24, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 122, 123, 124, 23, 12, and 24, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 32, 33, 34, 12 and 35, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 38, 39, 40 and 41, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 38, 49, 50 and 51, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 42, 43 11, 12 and 13, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 44, 45, 46, 29 and 47, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 52, 49, 50 and 51, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 53, 54, 55, 23, 12 and 35, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 29, and 30, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 131, 29, and 132, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 133, and 132, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29, and 30, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 131, 29, and 132, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29, and 136, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29, and 30, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 131, 29, and 132, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29, and 136, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 131, 29, and 132, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 133, and 132, respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 137, 27, 28, 133, and 132, respectively. In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises a heavy chain variable region (VH) of SEQ ID NOs: 60, 62, 64, 66, 68, 70, 72, 74, 75, 77, 79, 160, 138, 139, or 140.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises a light chain variable region (VL) or SEQ ID NOs: 61, 63, 65, 67, 69, 71, 73, 76, 78, 142, 143, or 144.

The VH, the VL, the HCDR and the LCDR sequences of exemplary antibodies specifically binding PSMA of the invention are shown in Table 2.

Table 2 provides a summary of examples of some PSMA-specific antibodies described herein:

TABLE 2 CDR sequences (as defined by Kabat) of mAbs   generated from phage panning against human PSMA (SEQ ID NO:) CDRs (SEQ ID NO:) FAB ID CDR1 CDR2 CDR3 PSMB129 HC NAWIS WINPESGRA ELYYLVYSTY (8) NYAQKFQG YYAFDY (9) (10) LC RASQSIDRWLN AASSLQS QQSPRYPLT (11) (12) (13) PSMB130 HC SYDIS GIIPIEGTA DYPAGYGFDY (14) NYAQKFQG (16) (15) LC RASQSVSSSYLA GASSRAT QQYGSSPLT (17) (18) (19) PSMB128 HC SDWMS AISGNGGST DPYYYDGDSY (20) EYADSVKG YGMDV (21) (22) LC RASQSISSYLN AASSLQS QQSYSTP (23) (12) (24) PSMB127 HC SDAMH EISGSGGYT DSYDSSLYVG (25) NYADSVKG DYFDY (26) (27) LC RASQSVSSYLA DASNRAT QQRSNWPLT (28) (29) (30) PSMB124 HC SYAIS WISPYNGNA DSDRSYNLDY (31) NYAQKFQG (33) (32) LC RASQSISGWLN AASSLQS QQSYSTPLT (34) (12) (35) PSMB123 HC SYWIG IIYPGDSDT GLPIWYLDY (36) RYSPSFQG (38) (37) LC RASQSVASDLA FASNRAT QQSITWPFT (39) (40) (41) PSMB122 HC SYAIS WIIPYNGNA VNSAALVWE (31) NYAQKFQG RLDY (42) (43) LC RASQSIDRWLN AASSLQS QQSPRYPLT (11) (12) (13) PSMB123 HC SYAIS GIIPIFGTA ASRVWHASY (31) NYAQKFQG GYLDY (44) (45) LC RASQSVSKWLA DASNRAT QQRFTAPWT (46) (29) (47) PSMB120 HC SYWIG IIYPGDSDT GWAYDRGLDY (36) RYSPSFQG (48) (37) LC KSSQSVLYSSN WASTRES QQYYSTPLT NKNYLA (50) (51) (49) PSMB119 HC SYWIG IIYPGDSDT AYHYSKGLDY (36) RYSPSFQG (52) (37) LC KSSQSVLYSSN WASTRES QQYYSTPLT NKNYLA (50) (51) (49) PSMB126 HC DYAIS RIDPIEGTA DRYYYDGVYW (53) NYAQKFQG YSDYFDY (54) (55) LC RASQSISSYLN AASSLQS QQSYSTPLT (23) (12) (35) PSMB87 HC SYWIS IITPGDSYT DYEWELFDSR (122) RYSPSFQG LDY (123) (124) LC RASQSISSYLN AASSLQS QQSYSTP (23) (12) (24) PSMB344 HC SDAMH EISGSGGYT DSYDSSLYVG (25) NYADSMKG DYFDY (130) (27) LC RASQSVSSYLA DASNRAT QQRSNWPLT (28) (29) (30) PSMB345 HC SDAMH EISGSGGYT DSYDSSLYVG (25) NYADSMKG DYFDY (130) (27) LC RASQSVSNYLA DASNRAT QQRRNWPLT (131) (29) (132) PSMB346 HC SDAMH EISGSGGYT DSYDSSLYVG (25) NYADSMKG DYFDY (130) (27) LC RASQSVSSYLA DASYRAT QQRRNWPLT (28) (133) (132) PSMB347 HC SDAMH EISGSGGYT DSYDSSLYVG (25) NYADSMKS DYFDY (134) (27) LC RASQSVSSYLA DASNRAT QQRSNWPLT (28) (29) (30) PSMB349 HC SDAMH EISGSGGYT DSYDSSLYVG (25) NYADSLKG DYFDY (135) (27) LC RASQSVSSYLA DASNRAT QQRGNWPLT (28) (29) (136) PSMB358 HC SDAMH EISGSGGYT DSYDSSLYVG (25) NYADSLKG DYFDY (135) (27) LC RASQSVSSYLA DASNRAT QQRSNWPLT (28) (29) (30) PSMB359 HC SDAMH EISGSGGYT DSYDSSLYVG (25) NYADSLKG DYFDY (135) (27) LC RASQSVSNYLA DASNRAT QQRRNWPLT (131) (29) (132) PSMB360 HC SDAMH EISGSGGYT DSYDSSLYVG (25) NYADSLKG DYFDY (135) (27) LC RASQSVSSYLA DASYRAT QQRRNWPLT (28) (133) (132) PSMB361 HC SDAMH EISGSGGYT DSYDSSLYVG (25) NYADSMKS DYFDY (134) (27) LC RASQSVSSYLA DASNRAT QQRGNWPLT (28) (29) (136) PSMB362 HC SDAMH EISGSGGYT DSYDSSLYVG (25) NYADSMKS DYFDY (134) (27) LC RASQSVSNYLA DASNRAT QQRRNWPLT (131) (29) (132) PSMB363 HC SDAMH EISGSGGYT DSYDSSLYVG (25) NYADSMKS DYFDY (134) (27) LC RASQSVSSYLA DASYRAT QQRRNWPLT (28) (133) (132) PSMB365 HC SDAMH EISGSGGYT DSYDSSLYVG (25) NYADSLKS DYFDY (137) (27) LC RASQSVSSYLA DASYRAT QQRRNWPLT (28) (133) (132)

In some embodiments are provided a PSMA-specific antibody, or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 2. In some embodiments are provided a PSMA-specific antibody, or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 2 and a light chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 2. In some embodiments described herein, the PSMA-specific antibody or antigen-binding fragment thereof competes for binding to PSMA with an antibody or antigen-binding comprising a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 2 and a light chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 2.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 14, 15, 16, 17, 18 and 19, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 62 and the VL of SEQ ID NO: 63.

In some embodiments, the antibody comprises a heavy chain (HC) having of SEQ ID NO: 84 and a light chain (LC) of SEQ ID NO: 85.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 20, 21, 22, 23, 12 and 24, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 64 and the VL of SEQ ID NO: 65.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 86 and a light chain (LC) of SEQ ID NO: 87.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 42, 43, 11, 12 and 13, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 74 and the VL of SEQ ID NO: 61.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 96 and a light chain (LC) of SEQ ID NO: 83.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 25, 26, 27, 28, 29 and 30, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 66 and the VL of SEQ ID NO: 67.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 88 and a light chain (LC) of SEQ ID NO: 89.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 122, 123, 124, 23, 12, and 24, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 160 and the VL of SEQ ID NO: 65.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 125 and a light chain (LC) of SEQ ID NO: 91.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 8, 9, 10, 11, 12, and 13, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 60 and the VL of SEQ ID NO: 61.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 82 and a light chain (LC) of SEQ ID NO: 83.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 31, 32, 33, 34, 12, and 35, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 70 and the VL of SEQ ID NO: 71.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 92 and a light chain (LC) of SEQ ID NO: 93.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 36, 37, 38, 39, 40, and 41, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 72 and the VL of SEQ ID NO: 73.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 94 and a light chain (LC) of SEQ ID NO: 95.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 31, 44, 45, 46, 29, and 47, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 75 and the VL of SEQ ID NO: 76.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 97 and a light chain (LC) of SEQ ID NO: 98.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 36, 37, 48, 49, 50, and 51, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 77 and the VL of SEQ ID NO: 78.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 99 and a light chain (LC) of SEQ ID NO: 100.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 36, 37, 52, 49, 50, and 51, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 79 and the VL of SEQ ID NO: 78.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 101 and a light chain (LC) of SEQ ID NO: 100.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 53, 54, 55, 23, 12, and 35, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 68 and the VL of SEQ ID NO: 69.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 90 and a light chain (LC) of SEQ ID NO: 91.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 25, 130, 27, 28, 29, and 30, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 138 and the VL of SEQ ID NO: 67.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 145 and a light chain (LC) of SEQ ID NO: 89.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 25, 130, 27, 131, 29, and 132, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 138 and the VL of SEQ ID NO: 142.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 145 and a light chain (LC) of SEQ ID NO: 148.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 25, 130, 27, 28, 133, and 132, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 138 and the VL of SEQ ID NO: 143.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 145 and a light chain (LC) of SEQ ID NO: 149.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 25, 134, 27, 28, 29, and 30, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 67.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 146 and a light chain (LC) of SEQ ID NO: 89.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 25, 135, 27, 28, 29, and 136, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 144.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 147 and a light chain (LC) of SEQ ID NO: 150.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 25, 135, 27, 28, 29, and 30, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 67.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 147 and a light chain (LC) of SEQ ID NO: 89.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 25, 135, 27, 131, 29, and 132, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 143.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 147 and a light chain (LC) of SEQ ID NO: 148.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 25, 135, 27, 28, 133, and 132, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 67.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 147 and a light chain (LC) of SEQ ID NO: 149.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 25, 134, 27, 28, 29, and 136, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 144.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 146 and a light chain (LC) of SEQ ID NO: 150.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 25, 134, 27, 131, 29, and 132, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 67.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 139 and a light chain (LC) of SEQ ID NO: 142.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 25, 134, 27, 28, 133, and 132, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 143.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 146 and a light chain (LC) of SEQ ID NO: 149.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs 25, 137, 27, 28, 133, and 132, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 141 and the VL of SEQ ID NO: 143.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 151 and a light chain (LC) of SEQ ID NO: 149.

In some embodiments, the antibody binds human PSMA ECD with an equilibrium dissociation constant (K_(D)) of less than about 100 nM, optionally less than about 50 nM, for example less than about 12 nM, wherein the K_(D) is measured using ProteOn XPR36 system at +25° C.

In some embodiments, the antibody binds cynomolgus PSMA ECD with an equilibrium dissociation constant (K_(D)) of less than about 100 nM, optionally less than about 50 nM, for example less than about 12 nM, wherein the K_(D) is measured using ProteOn XPR36 system at +25° C.

In some embodiments, the antibody is of IgG4 isotype, optionally comprising a heavy chain substitution S228P, F234A, and L235A when compared to the wild type IgG4.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 74 and the VL of SEQ ID NO: 61 and is of IgG4 isotype, optionally comprising a heavy chain substitution S228P, F234A, and L235A when compared to the wild type IgG4.

In some embodiments, the antibody is a multispecific antibody, such as a bispecific PSMA/CD3 antibody.

The antibody is suitable for use in therapy, for example in treating cancer.

The antibody is suitable for use in therapy, for example in treating a solid tumor.

The antibody is suitable for use in therapy, for example in treating a prostate cancer or castration-resistant prostate cancer.

The antibody is suitable for use in therapy, for example in treating a prostatic intraepithelial neoplasia.

The antibody is suitable for use in therapy, for example in treating colorectal cancer.

The antibody is suitable for use in therapy, for example in treating a clear cell renal carcinoma.

The antibody is suitable for use in therapy, for example in treating gastric cancer.

The antibody is suitable for use in therapy, for example in treating a renal cell carcinoma (RCC) (e.g., a kidney clear cell carcinoma or a kidney papillary cell carcinoma), or a metastatic lesion thereof.

The antibody is suitable for use in therapy, for example in treating a bladder cancer.

The antibody is suitable for use in therapy, for example in treating a breast cancer

The antibody is suitable for use in therapy, for example in treating a kidney cancer.

The antibody is suitable for use in therapy, for example in treating a neovascular disorder such as, for example, a cancer characterized by solid tumor growth.

The antibody is suitable for use in therapy, for example in treating a neovascular disorder such as, for example, clear cell renal carcinoma (CCRCC colorectal cancer, breast cancer, bladder cancer, lung cancer, and pancreatic cancer and various other non-prostate cancers, including but not limited to renal, urothelial, lung, colon, breast, and adenocarcinaoma to the liver.

The IgG class is divided in four isotypes: IgG1, IgG2, IgG3 and IgG4 in humans. They share more than 95% homology in the amino acid sequences of the Fc regions but show major differences in the amino acid composition and structure of the hinge region. The Fc region mediates effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to Fc receptors (FcgRs) on the surface of immune effector cells such as natural killers and macrophages, leading to the phagocytosis or lysis of the targeted cells. In CDC, the antibodies kill the targeted cells by triggering the complement cascade at the cell surface. The antibodies described herein include antibodies with the described features of the variable domains in combination with any of the IgG isotypes, including modified versions in which the Fc sequence has been modified to effect different effector functions.

For many applications of therapeutic antibodies, Fc-mediated effector functions are not part of the mechanism of action. These Fc-mediated effector functions can be detrimental and potentially pose a safety risk by causing off-mechanism toxicity. Modifying effector functions can be achieved by engineering the Fc regions to reduce their binding to FcgRs or the complement factors. The binding of IgG to the activating (FcgRI, FcgRIIa, FcgRIIIa and FcgRIIIb) and inhibitory (FcgRIIb) FcgRs or the first component of complement (C1q) depends on residues located in the hinge region and the CH2 domain. Mutations have been introduced in IgG1, IgG2 and IgG4 to reduce or silence Fc functionalities. The antibodies described herein may include these modifications.

In one embodiment, the antibody comprises an Fc region with one or more of the following properties: (a) reduced effector function when compared to the parent Fc; (b) reduced affinity to Fcg RI, Fcg RIIa, Fcg RIIb, Fcg RIIIb and/or Fcg RIIIa, (c) reduced affinity to FcgRI (d) reduced affinity to FcgRIIa (e) reduced affinity to FcgRIIb, (f) reduced affinity to Fcg RIIIb or (g) reduced affinity to FcgRIIIa.

In some embodiments, the antibodies or antigen-binding fragments are IgG, or derivatives thereof, e.g., IgG1, IgG2, IgG3, and IgG4 isotypes. In some embodiments wherein the antibody has an IgG4 isotype, the antibody contains S228P, L234A, and L235A substitutions in its Fc region. In some embodiments wherein the antibody has an IgG1 isotype, the antibody contains S228P, L234A, and L235A substitutions in its Fc region. The antibodies described herein may include these modifications. In some embodiments, the antibody has an IgG1 isotype

In some embodiments, the antibody is of IgG4 isotype, optionally comprising a heavy chain substitution S228P when compared to the wild type IgG4.

In some embodiments, the antibody is of IgG isotype, optionally comprising heavy chain substitutions L234A, G237A, P238S, H268A, V309L, A330S and P331 S when compared to the wild type IgG1.

In addition to the described PSMA-specific antibodies and antigen-binding fragments, also provided are polynucleotide sequences capable of encoding the described antibodies and antigen-binding fragments. Vectors comprising the described polynucleotides are also provided, as are cells expressing the PSMA-specific antibodies or antigen-binding fragments provided herein. Also described are cells capable of expressing the disclosed vectors. These cells may be mammalian cells (such as 293F cells, CHO cells), insect cells (such as Sf7 cells), yeast cells, plant cells, or bacteria cells (such as E. coli). The described antibodies may also be produced by hybridoma cells.

Homologous Antibodies

Variants of the antibodies specifically binding PSMA of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, comprising the VH, the VL or the VH and the VL amino acid sequences shown in Table 3 are within the scope of the invention. For example, variants may comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions in the VH and/or the VL as long as the homologous antibodies retain or have improved functional properties when compared to the parental antibodies. In some embodiments, the sequence identity may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to a VH or the VL amino acid sequence of the invention. Optionally, any variation of the variant compared to the parental antibody is not within the CDRs of the variant.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NOs: 60, 62, 64, 66, 68, 70, 72, 74, 75, 77, 79, 160, 138, 139, or 140, the VH optionally having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VL of SEQ ID NOs: 61, 63, 65, 67, 69, 71, 73, 76, 78, 142, 143, or 144, the VL optionally having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions.

Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 60 and the VL of SEQ ID NO: 61, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 62 and the VL of SEQ ID NO: 63, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 64 and the VL of SEQ ID NO: 65, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 66 and the VL of SEQ ID NO: 67, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 68 and the VL of SEQ ID NO: 69, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 70 and the VL of SEQ ID NO: 71, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 72 and the VL of SEQ ID NO: 73, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 74 and the VL of SEQ ID NO: 61, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 75 and the VL of SEQ ID NO: 76, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 77 and the VL of SEQ ID NO: 78, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 79 and the VL of SEQ ID NO: 78, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 160 and the VL of SEQ ID NO: 65, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 138 and the VL of SEQ ID NO: 67, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 138 and the VL of SEQ ID NO: 142, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 138 and the VL of SEQ ID NO: 143, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 67, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 144, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 67, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 142, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 143, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 144, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 142, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 143, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH of SEQ ID NO: 141 and the VL of SEQ ID NO: 143, wherein the VH, the VL or both the VH and the VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitutions are not within the CDRs.

The homologous antibodies specifically binding PSMA of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, have one, two, three, four or five of the following properties:

-   -   a) binds Pan troglodyte PSMA ECD with an equilibrium         dissociation constant (K_(D)) of 25 nM or less, wherein the         K_(D) is measured using ProteOn XPR36 system at +25° C.,     -   b) binds LNCaP cells with a calculated EC₅₀ of 20 nM or less and         binds Macaca fascicularis PSMA-expressing HEK cells with a         calculated EC₅₀ of 40 nM or less, wherein the difference in         calculated EC₅₀ between binding LNCaP cells and binding Macaca         fascicularis PSMA-expressing HEK cells is less than 5-fold, and         wherein the calculated EC₅₀ is measured in a whole cell binding         assay at 0° C. using flow cytometry,     -   c) binds recombinant PSMA ECD from human (SEQ ID NO: 7), Pan         troglodytes (SEQ ID NO:4) and Macaca fascicularis (SEQ ID NO: 5)         with an equilibrium dissociation constant (K_(D)) of 12 nM or         less, wherein the K_(D) is measured using Proteon surface         plasmon resonance assay ProteOn XPR36 system at +25C;     -   d) displays T-cell mediated killing of LNCaP cells, C42 cells,         human PSMA-expressing HEK cells or Macaca fascicularis         PSMA-expressing HEK cells when paired in a bispecific antibody         with anti-CD3 antibody CD3B219, wherein the T-cell mediated         killing is measured by Chromium-51 or by caspase 3/7 activation         assay or     -   e) recognizes a conformational epitope wherein the epitope is         comprised of residues 1138, F235, P237, G238, D244, Y299, Y300,         Q303, K304, E307, and K324-P326 of human PSMA (SEQ ID NO:3)         Antibodies with Conservative Modifications

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the VH comprising the HCDR1, the HCDR2 and the HCDR3 sequences and the VL comprising the LCDR1, the LCDR2 and the LCDR3 sequences, wherein one or more of the CDR sequences comprise specified amino acid sequences based on the antibodies described herein (e.g., antibodies shown in Table 2, or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the parental antibodies specifically binding PSMA of the invention.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 8, 9, 10, 11, 12 and 13, respectively, and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 14, 15, 16, 17, 18 and 19, respectively, and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 20, 21, 22, 23, 12 and 24, respectively, and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively, and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 32, 33, 34, 12 and 35, respectively, and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 38, 39, 40 and 41, respectively, and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 42, 43 11, 12 and 13, respectively, and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 44, 45, 46, 29 and 47, respectively, and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 48, 49, 50 and 51, respectively, and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 52, 49, 50 and 51, respectively, and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 53, 54, 55, 23, 12 and 35, respectively, and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 29, and 30, respectively and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 131, 29, and 132, respectively and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 133, and 132, respectively and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29, and 30, respectively and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29, and 136, respectively and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29, and 30, respectively and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 131, 29, and 132, respectively and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 131, 29, and 132, respectively and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29, and 136, respectively and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 131, 29, and 132, respectively and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 133, and 132, respectively and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 137, 27, 28, 133, and 132, respectively and conservative modifications thereof.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 122, 123, 124, 23, 12 and 24, respectively, and conservative modifications thereof.

The antibodies with conservative modifications of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, have one, two, three, four or five of the following properties:

-   -   a) binds Pan troglodyte PSMA ECD with an equilibrium         dissociation constant (K_(D)) of 25 nM or less, wherein the Kr         is measured using ProteOn XPR36 system at +25C,     -   b) binds LNCaP cells with a calculated EC₅₀ of 20 nM or less and         binds Macaca fascicularis PSMA-expressing HEK cells with a         calculated EC₅₀ of 40 nM or less, wherein the difference in         calculated EC₅₀ between binding LNCaP cells and binding Macaca         fascicularis PSMA-expressing HEK cells is less than 5-fold, and         wherein the calculated EC₅₀ is measured in a whole cell binding         assay at 0° C. using flow cytometry,     -   c) binds recombinant PSMA ECD from human(SEQ ID NO: 7), Pan         troglodytes (SEQ ID NO:4) and Macaca fascicularis (SEQ ID NO: 5)         with an equilibrium dissociation constant (K_(D)) of 12 nM or         less, wherein the K_(D) is measured using Proteon surface         plasmon resonance assay ProteOn XPR36 system at +25° C.;     -   d) displays T-cell mediated killing of LNCaP cells, C42 cells,         human PSMA-expressing HEK cells or Macaca fascicularis         PSMA-expressing HEK cells when paired in a bispecific antibody         with anti-CD3 antibody CD3B219, wherein the T-cell mediated         killing is measured by Chromium-51 or by caspase 3/7 activation         assay or     -   e) recognizes a conformational epitope wherein the epitope is         comprised of residues 1138, F235, P237, G238, D244, Y299, Y300,         Q303, K304, E307, and K324-P326 of human PSMA (SEQ ID NO:3)

“Conservative modifications” refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequences. Conservative modifications include amino acid substitutions, additions and deletions. Conservative substitutions are those in which the amino acid is replaced with an amino acid residue having a similar side chain. The families of amino acid residues having similar side chains are well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amide (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine) and sulfur-containing side chains (cysteine, methionine). Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643:55-67, Sasaki et al., (1988) Adv Biophys 35:1-24). Amino acid substitutions to the antibodies of the invention may be made by known methods for example by PCR mutagenesis (U.S. Pat. No. 4,683,195). Alternatively, libraries of variants may be generated for example using random (NNK) or non-random codons, for example DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting antibody variants may be tested for their characteristics using assays described herein.

Immunoconjugates

An “immunoconjugate” refers to the antibody of the invention conjugated to one or more heterologous molecule(s).

In some embodiments, the antibody of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, is conjugated to one or more cytotoxic agents. Exemplary such cytotoxic agents include chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), and radioactive isotopes.

In some embodiments, an immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody of the invention is conjugated to one or more drugs, such as to a maytansinoid (see, e.g., U.S. Pat. Nos. 5,208,020, 5,416,06)); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see, e.g., U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298), a dolastatin, a calicheamicin or derivative thereof (see, e.g., U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739, 116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., (1993) Cancer Res 53:3336-3342; and Lode et al., (1998) Cancer Res 58:2925-2928); an anthracycline such as daunomycin or doxorubicin (see, e.g., Kratz et al., (2006) Current Med. Chem 13:477-523; Jeffrey et al., (2006) Bioorganic & Med Chem Letters 16:358-362; Torgov et al., (2005) Bioconj Chem 16:717-721; Nagy et al., (2000) Proc Natl Acad Sci USA 97:829-834; Dubowchik et al, Bioorg. & Med. Chem. Letters 12: 1529-1532 (2002); King et al., (2002) J Med Chem 45:4336-4343; and U.S. Pat. No. 6,630,579), methotrexate, vindesine, a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel.

In some embodiments, the immunoconjugate comprises the antibody of the invention described herein conjugated to an enzymatically active toxin or fragment thereof, such as diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody is conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugates. Examples include At211, I131, I1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When the radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-I1, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of the antibody of the invention described herein and the cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HQ), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin may be prepared as described in Vitetta et al., (1987) Science 238: 1098. Carbon-14-labeled I-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See, e.g., WO94/11026. The linker may be a “cleavable linker” facilitating release of a cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., (1992) Cancer Res 52: 127-131; U.S. Pat. No. 5,208,020) may be used.

The immunoconjugates or ADCs may be prepared with cross-linker reagents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

One embodiment of the invention, and in some embodiments of each and every one of the numbered embodiments listed below, is an immunoconjugate comprising the antibody specifically binding PSMA of the invention linked to a therapeutic agent or an imaging agent.

Another embodiment of the invention, and in some embodiments of each and every one of the numbered embodiments listed below, is an immunoconjugate comprising the antibody specifically binding CD3 of the invention linked to a therapeutic agent or an imaging agent.

Another embodiment of the invention, and in some embodiments of each and every one of the numbered embodiments listed below, is an immunoconjugate comprising the bispecific PSMA/CD3 antibody of the invention linked to a therapeutic agent or an imaging agent.

Generation of Monospecific Antibodies of the Invention

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antagonistic antibodies specifically binding PSMA of the invention are human.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the antagonistic antibodies specifically binding PSMA of the invention are humanized.

Monospecific antibodies of the invention described herein (e.g. antibodies specifically binding PSMA) may be generated using various technologies. For example, the hybridoma method of Kohler and Milstein, Nature 256:495, 1975 may be used to generate monoclonal antibodies. In the hybridoma method, a mouse or other host animal, such as a hamster, rat or monkey, is immunized with human chimpanzee or macaque PSMA or CD3 or fragments of PSMA or CD3, such as the extracellular domain of PSMA or CD3, followed by fusion of spleen cells from immunized animals with myeloma cells using standard methods to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Colonies arising from single immortalized hybridoma cells are screened for production of antibodies with desired properties, such as specificity of binding, cross-reactivity or lack thereof, and affinity for the antigen.

Various host animals may be used to produce the PSMA antibodies of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below. For example, Balb/c mice may be used to generate mouse anti-human PSMA antibodies. The antibodies made in Balb/c mice and other non-human animals may be humanized using various technologies to generate more human-like sequences.

Exemplary humanization techniques including selection of human acceptor frameworks are known and include CDR grafting (U.S. Pat. No. 5,225,539), SDR grafting (U.S. Pat. No. 6,818,749), Resurfacing (Padlan, (1991) Mol Immunol 28:489-499), Specificity Determining Residues Resurfacing (U.S. Patent Publ. No. 2010/0261620), human framework adaptation (U.S. Pat. No. 8,748,356) or superhumanization (U.S. Pat. No. 7,709,226). In these methods, CDRs of parental antibodies are transferred onto human frameworks that may be selected based on their overall homology to the parental frameworks, based on similarity in CDR length, or canonical structure identity, or a combination thereof.

Humanized antibodies may be further optimized to improve their selectivity or affinity to a desired antigen by incorporating altered framework support residues to preserve binding affinity (backmutations) by techniques such as those described in Int. Patent Publ. Nos. WO1090/007861 and WO1992/22653, or by introducing variation at any of the CDRs for example to improve affinity of the antibody.

Transgenic animals, such as mice or rat carrying human immunoglobulin (Ig) loci in their genome may be used to generate human antibodies against a target protein, and are described in for example U.S. Pat. No. 6,150,584, Int. Patent Publ. No. WO99/45962, Int. Patent Publ. Nos. WO2002/066630, WO2002/43478, WO2002/043478 and WO1990/04036, Lonberg et al (1994) Nature 368:856-9; Green et al (1994) Nature Genet. 7:13-21; Green & Jakobovits (1998) Exp. Med. 188:483-95; Lonberg and Huszar (1995) Int Rev Immunol 13:65-93; Bruggemann et al., (1991) Eur J Immunol 21:1323-1326; Fishwild et al., (1996) Nat Biotechnol 14:845-851; Mendez et al., (1997) Nat Genet 15:146-156; Green (1999) J Immunol Methods 231:11-23; Yang et al., (1999) Cancer Res 59:1236-1243; Bruggemann and Taussig (1997) Curr Opin Biotechnol 8:455-458. The endogenous immunoglobulin loci in such animal may be disrupted or deleted, and at least one complete or partial human immunoglobulin locus may be inserted into the genome of the antimal using homologous or non-homologous recombination, using transchromosomes, or using minigenes. Companies such as Regeneron (http://_www_regeneron_com), Harbour Antibodies (http://_www_harbourantibodies_com), Open Monoclonal Technology, Inc. (OMT) (http://_www_omtinc_net), KyMab (http://_www_kymab_com), Trianni (http://_www.trianni_com) and Ablexis (http://_www_ablexis_com) may be engaged to provide human antibodies directed against a selected antigen using technologies as described above.

Human antibodies may be selected from a phage display library, where the phage is engineered to express human immunoglobulins or portions thereof such as Fabs, single chain antibodies (scFv), or unpaired or paired antibody variable regions (Knappik et al., (2000) J Mol Biol 296:57-86; Krebs et al., (2001) J Immunol Meth 254:67-84; Vaughan et al., (1996) Nature Biotechnology 14:309-314; Sheets et al., (1998) PITAS (USA) 95:6157-6162; Hoogenboom and Winter (1991) J Mol Biol 227:381; Marks et al., (1991) J Mol Biol 222:581). The antibodies of the invention may be isolated for example from phage display library expressing antibody heavy and light chain variable regions as fusion proteins with bacteriophage pIX coat protein as described in Shi et al., (2010) J Mol Biol 397:385-96, and Int. Patent Publ. No. WO09/085462). The libraries may be screened for phage binding to human and/or cyno PSMA or CD3 and the obtained positive clones may be further characterized, the Fabs isolated from the clone lysates, and expressed as full length IgGs. Such phage display methods for isolating human antibodies are described in for example: U.S. Pat. Nos. 5,223,409, 5,403,484, 5,571,698, 5,427,908, 5,580,717, 5,969,108, 6,172,197, 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081.

Preparation of immunogenic antigens and monoclonal antibody production may be performed using any suitable technique, such as recombinant protein production. The immunogenic antigens may be administered to an animal in the form of purified protein, or protein mixtures including whole cells or cell or tissue extracts, or the antigen may be formed de novo in the animal's body from nucleic acids encoding said antigen or a portion thereof.

Generation of Multispecific PSMA×CD3 Antibodies of the Invention

The multispecific PSMA×CD3 antibodies of the invention (e.g. the bispecific antibodies comprising a first domain specifically binding PSMA and a second domain specifically binding CD3) may be generated by combining PSMA binding VH/VL domains with CD3 binding VH/VL domains isolated and characterized herein. Alternatively, the bispecific PSMA×CD3 antibodies may be engineered using VH/VL domains from publicly available monospecific anti-PSMA and anti-CD3 antibodies, and/or by mix-matching the PSMA or CD3 binding VH/VL domains identified herein with publicly available PSMA or CD3 binding VH/VL domains.

Exemplary anti-PSMA antibodies that may be used to engineer bispecific PSMA×CD3 molecules are for example those herein and in Table 2. For example, the VH/VL domains of the PSMA antibodies of the invention may be incorporated into bispecific antibodies comprising CD3 binding VH/VL domains described herein and in Table 5. For example, the VH/VL domains of the CD3 antibodies CD3B217 and CD3B219 described herein may be used to generate bispecific PSMA×CD3 antibodies. In addition to the description and characterization of antibodies CD3B217 and CD3B219 provided herein, a more detailed description of the antibodies may be found in U.S. patent application publication number 2016-0068605 A1, which is incorporated by reference herein.

Similarly, exemplary anti-CD3 antibodies that may be used to engineer bispecific PSMA×CD3 molecules are for example those described in Int. Patent Publ. Nos. WO2005/048935, WO2004/106380 and WO2015095392. These CD3 VH/VL domains may be incorporated into bispecific antibodies comprising PSMA binding VH/VL domains described herein and in Table 2. For example, the VH/VL domains of the PSMA antibodies PSMB119, PSMB120, PSMB121, PSMB122, PSMB123, PSMB87, PSMB126, PSMB127, PSMB128, PSMB129, PSMB130, PSMB120, PSMB121, PSMB122, PSMB123, PSMB127, PSMB128, PSMB130, PSMB344, PSMB345, PSMB346, PSMB347, PSMB349, PSMB358, PSMB359, PSMB360, PSMB361, PSMB362, PSMB363, and PSMB365 described herein may be used to generate bispecific PSMA×CD3 antibodies.

The generated bispecific PSMA×CD3 antibodies may be tested for their binding to PSMA and CD3, and for their desired functional characteristics, such as T-cell mediated killing of PSMA-expressing cells (eg, LNCaP).

Bispecific antibodies of the invention comprise antibodies having a full length antibody structure.

“Full length antibody” refers to an antibody having two full length antibody heavy chains and two full length antibody light chains. A full length antibody heavy chain (HC) consists of well-known heavy chain variable and constant domains VH, CH1, hinge, CH2, and CH3. A full length antibody light chain (LC) consists of well-known light chain variable and constant domains VL and CL. The full length antibody may be lacking the C-terminal lysine (K) in either one or both heavy chains.

“Fab-arm” or “half molecule” refers to one heavy chain-light chain pair that specifically binds an antigen.

Full length bispecific antibodies of the invention described herein and in some embodiments of each and every one of the numbered embodiments listed below, may be generated for example using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies by introducing substitutions at the heavy chain CH3 interface in each half molecule to favor heterodimer formation of two antibody half molecules having distinct specificity either in vitro in cell-free environment or using co-expression. The Fab arm exchange reaction is the result of a disulfide-bond isomerization reaction and dissociation-association of CH3 domains. The heavy chain disulfide bonds in the hinge regions of the parental monospecific antibodies are reduced. The resulting free cysteines of one of the parental monospecific antibodies form an inter heavy-chain disulfide bond with cysteine residues of a second parental monospecific antibody molecule and simultaneously CH3 domains of the parental antibodies release and reform by dissociation-association. The CH3 domains of the Fab arms may be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody having two Fab arms or half molecules which each bind a distinct epitope, i.e. an epitope on PSMA and an epitope on CD3.

“Homodimerization” refers to an interaction of two heavy chains having identical CH3 amino acid sequences. “Homodimer” refers to an antibody having two heavy chains with identical CH3 amino acid sequences.

“Heterodimerization” refers to an interaction of two heavy chains having non-identical CH3 amino acid sequences. “Heterodimer” refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.

In some embodiments, the bispecific antibodies include designs such as the Triomab/Quadroma (Trion Pharma/Fresenius Biotech), Knob-in-Hole (Genentech), CrossMAbs (Roche) and the electrostatically-matched (Chugai, Amgen, NovoNordisk, Oncomed), the LUZ-Y (Genentech), the Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), the Biclonic (Merus) and the DuoBody (Genmab A/S).

The Triomab quadroma technology may be used to generate full length bispecific antibodies of the invention. Triomab technology promotes Fab arm exchange between two parental chimeric antibodies, one parental mAb having IgG2a and the second parental mAb having rat IgG2b constant regions, yielding chimeric bispecific antibodies.

The “knob-in-hole” strategy (see, e.g., Intl. Publ. No. WO 2006/028936) may be used to generate full length bispecific antibodies of the invention. Briefly, selected amino acids forming the interface of the CH3 domains in human IgG can be mutated at positions affecting CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into a heavy chain of an antibody specifically binding a first antigen and an amino acid with a large side chain (knob) is introduced into a heavy chain of an antibody specifically binding a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction of the heavy chain with a “hole” with the heavy chain with a “knob”. Exemplary CH3 substitution pairs forming a knob and a hole are (expressed as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T, T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S L368A_Y407V.

The CrossMAb technology may be used to generate full length bispecific antibodies of the invention. CrossMAbs, in addition to utilizing the “knob-in-hole” strategy to promoter Fab arm exchange, have in one of the half arms the CH1 and the CL domains exchanged to ensure correct light chain pairing of the resulting bispecific antibody (see e.g. U.S. Pat. No. 8,242,247).

Other cross-over strategies may be used to generate full length bispecific antibodies of the invention by exchanging variable or constant, or both domains between the heavy chain and the light chain or within the heavy chain in the bispecific antibodies, either in one or both arms. These exchanges include for example VH-CH1 with VL-CL, VH with VL, CH3 with CL and CH3 with CH1 as described in Int. Patent Publ. Nos. WO2009/080254, WO2009/080251, WO2009/018386 and WO2009/080252.

Other strategies such as promoting heavy chain heterodimerization using electrostatic interactions by substituting positively charged residues at one CH3 surface and negatively charged residues at a second CH3 surface may be used, as described in US Patent Publ. No. US2010/0015133; US Patent Publ. No. US2009/0182127; US Patent Publ. No. US2010/028637 or US Patent Publ. No. US2011/0123532. In other strategies, heterodimerization may be promoted by following substitutions (expressed as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): L351Y_F405A_Y407V/T394W, T366I_K392M_T394W/F405A_Y407V, T366L_K392M_T394W/F405A_Y407V, L351Y_Y407A/T366A_K409F, L351Y_Y407A/T366V_K409F, Y407A/T366A_K409F, or T350V L351Y_F405A_Y407V/T350V T366L_K392L T394W as described in U.S. Patent Publ. No. US2012/0149876 or U.S. Patent Publ. No. US2013/0195849.

LUZ-Y technology may be utilized to generate bispecific antibodies of the invention. In this technology, a leucine zipper is added into the C terminus of the CH3 domains to drive the heterodimer assembly from parental mAbs that is removed post-purification as described in Wranik et al., (2012) J Biol Chem 287(52): 42221-9.

SEEDbody technology may be utilized to generate bispecific antibodies of the invention. SEEDbodies have, in their constant domains, select IgG residues substituted with IgA reisdues to promote heterodimerszation as described in U.S. Patent No. US20070287170.

The present invention also provides for a multispecific, multifunctional antibody that specifically binds to PSMA.

According to the invention such a multispecific, multifunctional antibody that specifically binds to PSMA may be a trispecific antibody for dual targeting of tumor cells—these are trifunctional structures that can be designed to target two different targets/epitopes on the tumor cell and with the third functionality bind with high affinity to either T-cells or NK-cells. Trispecific antibodies targeting two distinct tumor epitopes and engaging T- or NK-cells lyse the tumor cells that express both targets. Such molecules can be generated by antibody formats known in the art and are fully described. (WO20151842071, WO2015158636, WO2010136172, WO2013174873). In a trispecific embodiment of the invention the antigen-binding polypeptide is bispecific for PSMA and a second distinct antigen on a tumor cell and additionally specific for an effector cell, in particular a T cell or a NK cell.

Bispecific antibodies of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, may be generated in vitro in a cell-free environment by introducing asymmetrical mutations in the CH3 regions of two monospecific homodimeric antibodies and forming the bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies in reducing conditions to allow disulfide bond isomerization according to methods described in Int.Patent Publ. No. WO2011/131746 (DuoBody technology). In the methods, the first monospecific bivalent antibody (e.g., anti-PSMA antibody) and the second monospecific bivalent antibody (e.g., anti-CD3 antibody) are engineered to have certain substitutions at the CH3 domain that promoter heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; thereby generating the bispecific antibody by Fab arm exchange. The incubation conditions may optimally be restored to non-reducing. Exemplary reducing agents that may be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercaptoethanol. For example, incubation for at least 90 min at a temperature of at least 20° C. in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH of from 5-8, for example at pH of 7.0 or at pH of 7.4 may be used.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the isolated bispecific antibody comprising a first domain specifically binding PSMA and a second domain specifically binding CD3 comprises at least one substitution in an antibody CH3 constant domain.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the at least one substitution in the antibody CH3 constant domain is 409R, F405L or F405L and R409K substitution, wherein residue numbering is according to the EU Index.

Antibody domains and numbering are well known. “Asymmetrical” refers to non-identical substitutions in the two CH3 domains in two separate heavy chains in an antibody. An IgG1 CH3 region typically consists of residues 341-446 on IgG1 (residue numbering according to the EU index).

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the isolated bispecific PSMA×CD3 antibody comprises a F405L substitution in an antibody first heavy chain (HC1) and a 409R substitution in an antibody second heavy chain (HC2).

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the isolated bispecific PSMA×CD3 antibody comprises a S228P substitution in the HC1 and S228P, F405L and R409K substitutions in the HC2, wherein the antibody is of IgG4 isotype.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the HC1 contains the first domain specifically binding PSMA and the HC2 contains the second domain specifically binding CD3.

In some embodiments described herein, the bispecific antibody of the invention comprises at least one, two, three, four, five, six, seven or eight asymmetrical substitutions in the HC1 and the HC2 at residue positions 350, 366, 368, 370, 399, 405, 407 or 409, when residue numbering is according to the EU index.

In some embodiments described herein, the bispecific antibody of the invention comprises at least one, two, three or four asymmetrical substitutions in the HC1 and the HC2 at residue positions 350, 370, 405 or 409, when residue numbering is according to the EU index.

In some embodiments described herein, the bispecific antibody of the invention comprises at least one asymmetrical substitution in the HC1 and the HC2 at residue positions 405 or 409, when residue numbering is according to the EU index.

In some embodiments described herein, the bispecific antibody of the invention comprises a 409R or a F405L substitution in the HC1 and a 409R or a F405L substitution in the HC2, wherein residue numbering is according to the EU index.

In some embodiments described herein, the bispecific antibody of the invention comprises the F405L substitution in the HC1 and the 409R substitution in the HC2.

In some embodiments described herein, the bispecific antibody of the invention comprises at least one asymmetrical substitution in the HC1 and the HC2 at residue positions 366, 368, 370, 399, 405, 407 or 409, wherein residue numbering is according to the EU index.

In some embodiments described herein, the HC1 position 409 has an amino acid substitution other than Lys, Leu or Met and the HC2 position 405 has an amino acid substitution other than Phe.

In some embodiments described herein, the HC1 position 405 has an amino acid substitution other than Phe and the HC2 position 409 has an amino acid substitution other than Lys, Leu or Met.

In some embodiments described herein, the HC1 position 409 has an amino acid substitution other than Lys, Leu or Met and the HC2 position 405 has an amino acid substitution other than Phe, Arg or Gly.

In some embodiments described herein, the HC1 position 405 has an amino acid substitution other than Phe, Arg or Gly and the HC2 CH3 position 409 has an amino acid substitution other than Lys, Leu or Met

In some embodiments described herein, the HC1 CH3 has Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409 and the HC2 has an amino acid other than Phe at position 405 and a Lys at position 409.

In some embodiments described herein, the HC1 has an amino acid other than Phe at position 405 and Lys at position 409 and the HC2 has Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409.

In some embodiments described herein, the HC1 has Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409 and the HC2 has a substitution other than Phe, Arg or Gly at position 405 and Lys at position 409.

In some embodiments described herein, the HC1 has a substitution other than Phe, Arg or Gly at position 405 and Lys at position 409 and the HC2 has Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409.

In some embodiments described herein, the HC1 has Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409 and the HC2 has Leu at position 405 and Lys at position 409.

In some embodiments described herein, the HC1 has Leu at position 405 and Lys at position 409 and the HC2 has Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409.

In some embodiments described herein, the HC1 has Phe at position 405 and Arg at position 409 and the HC2 has an amino acid other than Phe, Arg or Gly at position 405 and Lys at position 409.

In some embodiments described herein, the HC has an amino acid other than Phe, Arg or Gly at position 405 and Lys at position 409 and the HC2 has Phe at position 405 and Arg at position 409.

In some embodiments described herein, the HC1 has Phe at position 405 and Arg at position 409 and the HC2 has Leu at position 405 and Lys at position 409.

In some embodiments described herein, the HC1 has Leu at position 405 and Lys at position 409 and the HC2 has Phe at position 405 and Arg at position 409.

In some embodiments described herein, the HC1 has Phe at position 405 and Lys at position 409 and the HC2 has Leu at position 405 and Arg at position 409.

In some embodiments described herein, the HC1 has Leu at position 405 and Arg at position 409 and the HC2 has Phe at position 405 and Lys at position 409.

In some embodiments described herein, the HC1 has an amino acid other than Lys, Leu or Met at position 409 and the HC2 has Lys at position 409, Thr at position 370 and Leu at position 405.

In some embodiments described herein, the HC1 has Lys at position 409, Thr at position 370 and Leu at position 405 and the HC2 has an amino acid other than Lys, Leu or Met at position 409.

In some embodiments described herein, the HC1 has Arg at position 409 and the HC2 has Lys at position 409, Thr at position 370 and Leu at position 405.

In some embodiments described herein, the HC1 has Lys at position 409, Thr at position 370 and Leu at position 405 and the HC2 has Arg at position 409.

In some embodiments described herein, the HC1 has Lys at position 370, Phe at position 405 and Arg at position 409 and the HC2 has Lys at position 409, Thr at position 370 and Leu at position 405.

In some embodiments described herein, the HC1 has Lys at position 409, Thr at position 370 and Leu at position 405 and the HC2 has Lys at position 370, Phe at position 405 and Arg at position 409.

In some embodiments described herein, the HC1 has an amino acid other than Lys, Leu or Met at position 409 and the HC2 has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gin, Arg, Ser or Thr at position 407.

In some embodiments described herein, the HC1 has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gin, Arg, Ser or Thr at position 407 and the HC2 has an amino acid other than Lys, Leu or Met at position 409.

In some embodiments described herein, the HC1 has an amino acid other than Lys, Leu or Met at position 409 and the HC2 has Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407.

In some embodiments described herein, the HC1 has Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and the HC2 has an amino acid other than Lys, Leu or Met at position 409.

In some embodiments described herein, the HC1 has an amino acid other than Lys, Leu or Met at position 409 and the HC2 has Gly, Leu, Met, Asn or Trp at position 407.

In some embodiments described herein, the HC1 has Gly, Leu, Met, Asn or Trp at position 407 and the HC2 has an amino acid other than Lys, Leu or Met at position 409.

In some embodiments described herein, the HC1 has Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409 and the HC2 has an amino acid other than Tyr, Asp. Glu, Phe, Lys, Gin, Arg, Ser or Thr at position 407 and Lys at position 409.

In some embodiments described herein, the HC1 has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gin, Arg, Ser or Thr at position 407 and Lys at position 409 and the HC2 has Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409.

In some embodiments described herein, the HC1 has Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409 and the HC2 has Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and Lys at position 409.

In some embodiments described herein, the HC1 has Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and Lys at position 409 and the HC2 CH3 has Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409.

In some embodiments described herein, the HC1 CH3 has Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409 and the HC2 has Gly, Leu, Met, Asn or Trp at position 407 and Lys at position 409.

In some embodiments described herein, the HC1 has Gly, Leu, Met, Asn or Trp at position 407 and Lys at position 409 and the HC2 has Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409.

In some embodiments described herein, the HC1 has Tyr at position 407 and Arg at position 409 and the HC2 has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gin, Arg, Ser or Thr at position 407 and Lys at position 409.

In some embodiments described herein, the HC1 has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gin, Arg, Ser or Thr at position 407 and Lys at position 409 and the HC2 has Tyr at position 407 and Arg at position 409.

In some embodiments described herein, the HC1 has Tyr at position 407 and Arg at position 409 and the HC2 has Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and Lys at position 409.

In some embodiments described herein, the HC1 has Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and Lys at position 409 and the HC2 has Tyr at position 407 and Arg at position 409.

In some embodiments described herein, the HC1 CH3 has Tyr at position 407 and Arg at position 409 and the HC2 CH3 has Gly, Leu, Met, Asn or Trp at position 407 and Lys at position 409.

In some embodiments described herein, the HC1 has Gly, Leu, Met, Asn or Trp at position 407 and Lys at position 409 and the HC2 has Tyr at position 407 and Arg at position 409.

In some embodiments described herein, the HC1 has an amino acid other than Lys, Leu or Met at position 409, and the HC2 has (i) an amino acid other than Phe, Leu and Met at position 368, or (ii) a Trp at position 370, or (iii) an amino acid other than Asp, Cys, Pro, Glu or Gin at position 399.

In some embodiments described herein, the HC1 has (i) an amino acid other than Phe, Leu and Met at position 368, or (ii) a Trp at position 370, or (iii) an amino acid other than Asp, Cys, Pro, Glu or Gin at position 399 and the HC2 has an amino acid other than Lys, Leu or Met at position 409.

In some embodiments described herein, the HC1 has Arg, Ala, His or Gly at position 409, and the HC2 has (i) Lys, Gin, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or (ii) Trp at position 370, or (iii) Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr at position 399.

In some embodiments described herein, the HC1 has (i) Lys, Gin, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or (ii) Trp at position 370, or (iii) Ala, Gly, lie, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr at position 399 and the HC2 has Arg, Ala, His or Gly at position 409.

In some embodiments described herein, the HC1 has Arg at position 409, and the HC2 has (i) Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or (ii) Trp at position 370, or (iii) Phe, His, Lys, Arg or Tyr at position 399.

In some embodiments described herein, the HC1 has (i) Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or (ii) Trp at position 370, or (iii) Phe, His, Lys, Arg or Tyr at position 399 and the HC2 has Arg at position 409.

In some embodiments described herein, the HC1 comprises a 409R substitution or a F405L substitution and the HC2 comprises a 409R substitution or a F405L substitution, wherein residue numbering is according to the EU index.

In some embodiments described herein, the HC1 comprises the F405L substitution and the HC2 comprises the 409R substitution.

Substitutions are typically made at the DNA level to a molecule such as the constant domain of the antibody using standard methods.

The antibodies of the invention may be engineered into various well-known antibody forms.

In some embodiments, the bispecific antibody of the present invention is a diabody or a cross-body.

In some embodiments, the bispecific antibodies include recombinant IgG-like dual targeting molecules, wherein the two sides of the molecule each contain the Fab fragment or part of the Fab fragment of at least two different antibodies; IgG fusion molecules, wherein full length IgG antibodies are fused to an extra Fab fragment or parts of Fab fragment; Fc fusion molecules, wherein single chain Fv molecules or stabilized diabodies are fused to heavy-chain constant-domains, Fc-regions or parts thereof; Fab fusion molecules, wherein different Fab-fragments are fused together; ScFv- and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) wherein different single chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g. domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.

In some embodiments, recombinant IgG-like dual targeting molecules include Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-one Antibody (Genentech) and mAb2 (F-Star).

In some embodiments, IgG fusion molecules include Dual Variable Domain (DVD)-Ig (Abbott), Ts2Ab (MedImmune/AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec) and TvAb (Roche).

In some embodiments, Fc fusion molecules include to ScFv/Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS) and Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics).

In some embodiments, Fab fusion bispecific antibodies include F(ab)2 (Medarex/AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech). ScFv-, diabody-based and domain antibodies include Bispecific T Cell Engager (BITE) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) and COMBODY (Epigen Biotech), dual targeting nanobodies (Ablynx), dual targeting heavy chain only domain antibodies. Various formats of bispecific antibodies have been described, for example in Chames and Baty (2009) Curr Opin Drug Disc Dev 12: 276 and in Nunez-Prado et al., (2015) Drug Discovery Today 20(5):588-594.

Polynucleotides, Vectors and Host Cells

Also disclosed are isolated polynucleotides that encode the antibodies or antigen-binding fragments that immunospecifically bind to PSMA. The isolated polynucleotides capable of encoding the variable domain segments provided herein may be included on the same, or different, vectors to produce antibodies or antigen-binding fragments.

Polynucleotides encoding recombinant antigen-binding proteins also are within the scope of the disclosure. In some embodiments, the polynucleotides described (and the peptides they encode) include a leader sequence. Any leader sequence known in the art may be employed. The leader sequence may include, but is not limited to, a restriction site or a translation start site.

The PSMA-specific antibodies or antigen-binding fragments described herein include variants having single or multiple amino acid substitutions, deletions, or additions that retain the biological properties (e.g., binding affinity or immune effector activity) of the described PSMA-specific antibodies or antigen-binding fragments. In the context of the present invention the following notations are, unless otherwise indicated, used to describe a mutation; i) substitution of an amino acid in a given position is written as e.g. K409R which means a substitution of a Lysine in position 409 with an Arginine; and ii) for specific variants the specific three or one letter codes are used, including the codes Xaa and X to indicate any amino acid residue. Thus, the substitution of Arginine for Lysine in position 409 is designated as: K409R, or the substitution of any amino acid residue for Lysine in position 409 is designated as K409X. In case of deletion of Lysine in position 409 it is indicated by K409*. Where a particular amino acid residue may vary among peptide isotypes or variants, and a substitution affects that residue in each isotype or variant or any of the isotypes or variants, the substitution is designated as, e.g., 409R, meaning the amino acid corresponding to position 409 is substituted with Arginine. The skilled person may produce variants having single or multiple amino acid substitutions, deletions, or additions.

These variants may include: (a) variants in which one or more amino acid residues are substituted with conservative or nonconservative amino acids. (b) variants in which one or more amino acids are added to or deleted from the polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the polypeptide is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the polypeptide, such as, for example, an epitope for an antibody, a polyhistidine sequence, a biotin moiety and the like. Antibodies or antigen-binding fragments described herein may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or nonconserved positions. In other embodiments, amino acid residues at nonconserved positions are substituted with conservative or nonconservative residues. The techniques for obtaining these variants, including genetic (deletions, mutations, etc.), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art.

The PSMA-specific antibodies or antigen-binding fragments described herein may embody several antibody isotypes, such as IgM, IgD, IgG, IgA and IgE. In some embodiments the antibody isotype is IgG1, IgG2, IgG3, or IgG4 isotype, preferably IgG1 or IgG4 isotype. Antibody or antigen-binding fragment thereof specificity is largely determined by the amino acid sequence, and arrangement, of the CDRs. Therefore, the CDRs of one isotype may be transferred to another isotype without altering antigen specificity. Alternatively, techniques have been established to cause hybridomas to switch from producing one antibody isotype to another (isotype switching) without altering antigen specificity. Accordingly, such antibody isotypes are within the scope of the described antibodies or antigen-binding fragments.

Also provided, are vectors comprising the polynucleotides described herein. The vectors can be expression vectors. Recombinant expression vectors containing a sequence encoding a polypeptide of interest are thus contemplated as within the scope of this disclosure.

The expression vector may contain one or more additional sequences such as but not limited to regulatory sequences (e.g., promoter, enhancer), a selection marker, and a polyadenylation signal. Vectors for transforming a wide variety of host cells are well known and include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), as well as other bacterial, yeast and viral vectors.

Recombinant expression vectors within the scope of the description include synthetic, genomic, or cDNA-derived nucleic acid fragments that encode at least one recombinant protein which may be operably linked to suitable regulatory elements. Such regulatory elements may include a transcriptional promoter, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. Expression vectors, especially mammalian expression vectors, may also include one or more nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5′ or 3′ flanking nontranscribed sequences, 5′ or 3′ nontranslated sequences (such as necessary ribosome binding sites), a polyadenylation site, splice donor and acceptor sites, or transcriptional termination sequences. An origin of replication that confers the ability to replicate in a host may also be incorporated.

The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells may be provided by viral sources. Exemplary vectors may be constructed as described by Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

In some embodiments, the antibody- or antigen-binding fragment-coding sequence is placed under control of a powerful constitutive promoter, such as the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin, human muscle creatine, and others. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use with the described embodiments. Such viral promoters include without limitation, Cytomegalovirus (CMV) immediate early promoter, the early and late promoters of SV40, the Mouse Mammary Tumor Virus (MMTV) promoter, the long terminal repeats (LTRs) of Maloney leukemia virus, Human Immunodeficiency Virus (HIV), Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV), and other retroviruses, and the thymidine kinase promoter of Herpes Simplex Virus. In one embodiment, the PSMA-specific antibody or antigen-binding fragment thereof coding sequence is placed under control of an inducible promoter such as the metallothionein promoter, tetracycline-inducible promoter, doxycycline-inducible promoter, promoters that contain one or more interferon-stimulated response elements (ISRE) such as protein kinase R 2′,5′-oligoadenylate synthetases, Mx genes, ADARI, and the like.

Vectors described herein may contain one or more Internal Ribosome Entry Site(s) (IRES). Inclusion of an IRES sequence into fusion vectors may be beneficial for enhancing expression of some proteins. In some embodiments the vector system will include one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the aforementioned nucleic acid sequences. Vector components may be contiguously linked, or arranged in a manner that provides optimal spacing for expressing the gene products (i.e., by the introduction of “spacer” nucleotides between the ORFs), or positioned in another way. Regulatory elements, such as the IRES motif, may also be arranged to provide optimal spacing for expression.

The vectors may comprise selection markers, which are well known in the art. Selection markers include positive and negative selection markers, for example, antibiotic resistance genes (e.g., neomycin resistance gene, a hygromycin resistance gene, a kanamycin resistance gene, a tetracycline resistance gene, a penicillin resistance gene), glutamate synthase genes, HSV-TK, HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase gene for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). A nucleic acid sequence encoding a selection marker or the cloning site may be upstream or downstream of a nucleic acid sequence encoding a polypeptide of interest or cloning site.

The vectors described herein may be used to transform various cells with the genes encoding the described antibodies or antigen-binding fragments. For example, the vectors may be used to generate PSMA-specific antibody or antigen-binding fragment-producing cells. Thus, another aspect features host cells transformed with vectors comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof that specifically binds PSMA, such as the antibodies or antigen-binding fragments described and exemplified herein.

Numerous techniques are known in the art for the introduction of foreign genes into cells and may be used to construct the recombinant cells for purposes of carrying out the described methods, in accordance with the various embodiments described and exemplified herein. The technique used should provide for the stable transfer of the heterologous gene sequence to the host cell, such that the heterologous gene sequence is heritable and expressible by the cell progeny, and so that the necessary development and physiological functions of the recipient cells are not disrupted. Techniques which may be used include but are not limited to chromosome transfer (e.g., cell fusion, chromosome mediated gene transfer, micro cell mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, microinjection, electroporation, liposome carrier), viral vector transfer (e.g., recombinant DNA viruses, recombinant RNA viruses) and the like (described in Cline, 29 Pharmac. Ther. 69-92 (1985)). Calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts with mammalian cells may also be used to transform cells.

Cells suitable for use in the expression of the PSMA-specific antibodies or antigen-binding fragments described herein are preferably eukaryotic cells, more preferably cells of plant, rodent, or human origin, for example but not limited to NSO, CHO, CHOK1, perC.6, Tk-ts13, BHK, HEK293 cells, COS-7, T98G, CV-1/EBNA, L cells, C127, 3T3, HeLa, NS1, Sp2/0 myeloma cells, and BHK cell lines, among others. In addition, expression of antibodies may be accomplished using hybridoma cells. Methods for producing hybridomas are well established in the art.

Cells transformed with expression vectors described herein may be selected or screened for recombinant expression of the antibodies or antigen-binding fragments described herein. Recombinant-positive cells are expanded and screened for subclones exhibiting a desired phenotype, such as high level expression, enhanced growth properties, or the ability to yield proteins with desired biochemical characteristics, for example, due to protein modification or altered post-translational modifications. These phenotypes may be due to inherent properties of a given subclone or to mutation. Mutations may be effected through the use of chemicals, UV-wavelength light, radiation, viruses, insertional mutagens, inhibition of DNA mismatch repair, or a combination of such methods.

Pharmaceutical compositions/Administration

The invention provides for pharmaceutical compositions comprising the antibodies of the invention described herein and a pharmaceutically acceptable carrier. For therapeutic use, the antibodies of the invention may be prepared as pharmaceutical compositions containing an effective amount of the antibody as an active ingredient in a pharmaceutically acceptable carrier. “Carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the antibody of the invention is administered. Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of the antibodies of the invention in such pharmaceutical formulation may vary, from less than about 0.5%, usually to at least about 1% to as much as 15 or 20% by weight and may be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g. Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, D. B. ed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.

The mode of administration for therapeutic use of the antibodies of the invention may be any suitable route that delivers the antibody to the host, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal), using a formulation in a tablet, capsule, solution, powder, gel, particle; and contained in a syringe, an implanted device, osmotic pump, cartridge, micropump; or other means appreciated by the skilled artisan, as well known in the art. Site specific administration may be achieved by for example intratumoral, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intracardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery.

The antibodies of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, may be administered to a subject by any suitable route, for example parentally by intravenous (i.v.) infusion or bolus injection, intramuscularly or subcutaneously or intraperitoneally. i.v. infusion may be given over for example 15, 30, 60, 90, 120, 180, or 240 minutes, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

The dose given to a subject is sufficient to alleviate or at least partially arrest the disease being treated (“therapeutically effective amount”) and may be sometimes 0.005 mg to about 100 mg/kg, e.g. about 0.05 mg to about 30 mg/kg or about 5 mg to about 25 mg/kg, or about 4 mg/kg, about 8 mg/kg, about 16 mg/kg or about 24 mg/kg, or for example about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but may even higher, for example about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg.

A fixed unit dose may also be given, for example, 50, 100, 200, 500 or 1000 mg, or the dose may be based on the patient's surface area, e.g., 500, 400, 300, 250, 200, or 100 mg/m2. Usually between 1 and 8 doses, (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) may be administered to treat the patient, but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses may be given.

The administration of the antibodies of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, may be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or longer. Repeated courses of treatment are also possible, as is chronic administration. The repeated administration may be at the same dose or at a different dose. For example, the antibodies of the invention described herein may be administered at 8 mg/kg or at 16 mg/kg at weekly interval for 8 weeks, followed by administration at 8 mg/kg or at 16 mg/kg every two weeks for an additional 16 weeks, followed by administration at 8 mg/kg or at 16 mg/kg every four weeks by intravenous infusion.

For example, the antibodies in the methods of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, may be provided as a daily dosage in an amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

The antibodies in the methods of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, may also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and/or reduce the risk of recurrence when a cancer is in remission.

The antibodies of the invention may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional protein preparations and well known lyophilization and reconstitution techniques can be employed.

Methods of Using PSMA-Specific Antibodies

PSMA is a prostate-cancer related cell membrane antigen frequently overexpressed in prostatic intraepithelial neoplasia (PIN), a condition in which some prostate cells have begun to look and behave abnormally; primary and metastatic prostate cancers; and the neovasculature of other solid tumors (e.g. breast, lung, bladder, kidney). PSMA expression correlates with disease progression and Gleason score. PSMA expression is increased in metastatic disease, hormone refractory cases, and higher-grade lesions, and it is further upregulated in androgen-insensitive tumors

Blockade of PSMA may inhibit or decrease the growth of PSMA-expressing cancerous cells and tumors in a subject. It may also have antiangiogenic activity owing to expression of PSMA in tumor neovasculature (Milowsky, et al. 2007). PSMA is highly expressed in prostatic intraepithelial neoplasia, the most established precursor of prostatic carcinoma, and therefore blockade of PSMA may modulate progression of PIN to prostate cancer

One embodiment of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below is a method of inhibiting growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of the antibody specifically binding PSMA of the invention.

Another embodiment of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below is a method of inhibiting the formation or growth of neovasculature of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of the antibody specifically binding PSMA of the invention.

Another embodiment of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below is a method of inhibiting progression of a precancerous state in a subject, comprising administering to the subject a therapeutically effective amount of the antibody specifically binding PSMA of the invention.

One embodiment of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below is a method of treating a cancer by administering to the subject in need thereof the antibody specifically binding PSMA of the invention described herein.

One embodiment of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below is a method of inhibiting growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of the bispecific antibody specifically binding PSMA×CD3 of the invention.

Another embodiment of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below is a method of inhibiting the formation or growth of neovasculature of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of the bispecific antibody specifically binding PSMA×CD3 of the invention.

Another embodiment of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below is a method of inhibiting progression of a precancerous state in a subject, comprising administering to the subject a therapeutically effective amount of the bispecific antibody specifically binding PSMA×CD3 of the invention.

One embodiment of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below is a method of treating a cancer by administering to the subject in need thereof the bispecific antibody specifically binding PSMA×CD3 of the invention.

Exemplary antibodies that may be used in the methods of the invention are antibodies specifically binding PSMA and bispecific PSMA×CD3 antibodies as described herein.

Exemplary PSMA antibodies that can be monospecific or can be part of a CD3 bispecific are antibodies PSMB19, PSMB120, PSMB121, PSMB122, PSMB123, PSMB87, PSMB126, PSMB127, PSMB128, PSMB129, PSMB130, PSMB120, PSMB121, PSMB122, PSMB123, PSMB127, PSMB128, PSMB130, PSMB344, PSMB345, PSMB346, PSMB347, PSMB349, PSMB358, PSMB359, PSMB360, PSMB361, PSMB362, PSMB363, and PSMB365, having the VH and the VL amino acid sequence and characteristics as described herein.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein is PSMB119, PSMB120, PSMB121, PSMB122, PSMB123, PSMB87, PSMB126, PSMB127, PSMB128, PSMB129, PSMB130, PSMB120, PSMB121, PSMB122, PSMB123, PSMB127, PSMB128, PSMB130, PSMB344, PSMB345, PSMB346, PSMB347, PSMB349, PSMB358, PSMB359, PSMB360, PSMB361, PSMB362, PSMB363, and PSMB365. The VH and the VL amino acid sequences of these antibodies are shown in Table 2.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 160 and the VL of SEQ ID NO: 65.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 79 and the VL of SEQ ID NO: 78.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 77 and the VL of SEQ ID NO: 78.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 75 and the VL of SEQ ID NO: 76.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 74 and the VL of SEQ ID NO: 61.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 72 and the VL of SEQ ID NO: 73.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 66 and the VL of SEQ ID NO: 67.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 64 and the VL of SEQ ID NO: 65.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 62 and the VL of SEQ ID NO: 63.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 60 and the VL of SEQ ID NO: 61

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 68 and the VL of SEQ ID NO: 69.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 70 and the VL of SEQ ID NO: 71.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 138 and the VL of SEQ ID NO: 67.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 138 and the VL of SEQ ID NO: 142.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 138 and the VL of SEQ ID NO: 143.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 62 and the VL of SEQ ID NO: 63.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 67.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 144.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 67.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 142.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 143.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 144.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 142.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 143.

In some embodiments, the antibody specifically binding PSMA used in the methods of the invention described herein comprises the VH of SEQ ID NO: 141 and the VL of SEQ ID NO: 143. In some embodiments, the bispecific PSMA/CD3 antibody comprising a first domain specifically binding PSMA and a second domain specifically binding CD3 used in the methods of the invention described herein, comprises the VH of SEQ ID NO: 62 and the VL of SEQ ID NO: 63 in the first domain, and the VH of SEQ ID NO: 104 and the VL of SEQ ID NO: 105 in the second domain.

In some embodiments, the bispecific PSMA/CD3 antibody comprising a first domain specifically binding PSMA and a second domain specifically binding CD3 used in the methods of the invention described herein, comprises the VH of SEQ ID NO: 64 and the VL of SEQ ID NO: 65 in the first domain, and the VH of SEQ ID NO: 104 and the VL of SEQ ID NO: 105 in the second domain.

In some embodiments, the bispecific PSMA/CD3 antibody comprising a first domain specifically binding PSMA and a second domain specifically binding CD3 used in the methods of the invention described herein, comprises the VH of SEQ ID NO: 66 and the VL of SEQ ID NO: 67 in the first domain, and the VH of SEQ ID NO: 104 and the VL of SEQ ID NO: 105 in the second domain.

In some embodiments, the bispecific PSMA/CD3 antibody comprising a first domain specifically binding PSMA and a second domain specifically binding CD3 used in the methods of the invention described herein, comprises the VH of SEQ ID NO: 75 and the VL of SEQ ID NO: 76 in the first domain, and the VH of SEQ ID NO: 104 and the VL of SEQ ID NO: 105 in the second domain.

In some embodiments, the bispecific PSMA/CD3 antibody comprising a first domain specifically binding PSMA and a second domain specifically binding CD3 used in the methods of the invention described herein, comprises the VH of SEQ ID NO: 74 and the VL of SEQ ID NO: 61 in the first domain, and the VH of SEQ ID NO: 104 and the VL of SEQ ID NO: 105 in the second domain.

In some embodiments, the bispecific PSMA/CD3 antibody comprising a first domain specifically binding PSMA and a second domain specifically binding CD3 used in the methods of the invention described herein, comprises the VH of SEQ ID NO: 160 and the VL of SEQ ID NO: 65 in the first domain, and the VH of SEQ ID NO: 104 and the VL of SEQ ID NO: 105 in the second domain.

Cancer may be a hyperproliferative condition or disorder, a solid tumor, a neovasculature, a soft tissue tumor, or a metastatic lesion.

“Cancer” is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathology type or stage of invasiveness. Examples of cancers include solid tumors, hematological malignancies, soft tissue tumors, and metastatic lesions. Exemplary solid tumors include malignancies, e.g., sarcomas, and carcinomas (including adenocarcinomas and squamous cell carcinomas) of the various organ systems, such as those affecting prostate, liver, lung, breast, lymphoid, gastrointestinal (e.g., colon), genitourinary tract (e.g., renal, urothelial cells), prostate and pharynx. Adenocarcinomas include malignancies such as most colon cancers, a rectal cancer, a renal-cell carcinoma, a liver cancer, a non-small cell carcinoma of the lung, a cancer of the small intestine and a cancer of the esophagus. Squamous cell carcinomas include malignancies, e.g., in the lung, esophagus, skin, head and neck region, oral cavity, anus, and cervix.

In one embodiment, the cancer is a prostate cancer.

Metastatic lesions of the aforementioned cancers may also be treated or prevented using the methods and antibodies of the invention described herein.

Exemplary cancers whose growth may be inhibited or reduced using the antibodies of the invention described herein include cancers that may overexpress PSMA. Exemplary such cancers include a prostate cancer or a prostatic intraepithelial neoplasia, a colorectal cancer, a gastric cancer, a clear cell renal carcinoma, a bladder cancer, a lung cancer, a squamous cell carcinoma, a glioma, a breast cancer, a kidney cancer, a neovascular disorder, a clear cell renal carcinoma (CCRCC), and a pancreatic cancer and various other non-prostate cancers, including but not limited to a renal cancer, a urothelial cancer and an adenocarcinaoma to the liver. Refractory or recurrent malignancies may be treated using the antibodies of the invention described herein.

Exemplary other cancers that may be treated with the antibodies of the invention described herein are anal cancer, a basal cell carcinoma, a biliary tract cancer, a bladder cancer, a bone cancer, brain and CNS cancers, a carcinoma of the fallopian tubes, carcinoma of the vagina, a carcinoma of the vulva, a cutaneous or intraocular malignant melanoma, a astro-esophageal cancer, a testicular cancer, an ovarian cancer, a pancreatic cancer, a rectal cancer, an uterine cancer, a primary CNS lymphoma; a neoplasm of the central nervous system (CNS), a cervical cancer, a choriocarcinoma, a rectum cancer, a connective tissue cancer, a cancer of the digestive system, an endometrial cancer, an eye cancer; an intra-epithelial neoplasm, a kidney cancer, a larynx cancer, a liver cancer; a small cell lung cancer, a neuroblastoma, an oral cavity cancer (e.g., lip, tongue, mouth, and pharynx), a nasopharyngeal cancer, a retinoblastoma, a rhabdomyosarcoma, a cancer of the respiratory system, a sarcoma, a thyroid cancer, a cancer of the urinary system, a hepatocarcinoma, a cancer of the anal region, a carcinoma of the fallopian tubes, a carcinoma of the vagina, a carcinoma of the vulva, a cancer of the small intestine, a cancer of the endocrine system, a cancer of the parathyroid gland, a cancer of the adrenal gland, a sarcoma of soft tissue, a cancer of the urethra, a cancer of the penis, solid tumors of childhood, a tumor angiogenesis, a spinal axis tumor, a brain stem glioma, a pituitary adenoma, Kaposi's sarcoma, Merkel cell cancer, an epidermoid cancer, a squamous cell cancer, an environmentally induced cancers including those induced by asbestos, as well as other carcinomas and sarcomas, and combinations of said cancers.

Patients having cancer including metastatic cancer that express PSMA may be treated with the antibodies of the invention described herein. The cancer may be a prostate cancer or a prostatic intraepithelial neoplasia, a colorectal cancer, a gastric cancer, a clear cell renal carcinoma, a bladder cancer, a lung cancer, a squamous cell carcinoma, a glioma, a breast cancer, a kidney cancer, a neovascular disorder, a clear cell renal carcinoma (CCRCC), and a pancreatic cancer and various other non-prostate cancers, including but not limited to a renal cancer, a urothelial cancer and an adenocarcinaoma to the liver.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the subject has a solid tumor.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the subject has a prostate tumor.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is a colorectal cancer.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is a gastric cancer.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is a lung cancer.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is a bladder cancer.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is a squamous cell carcinoma.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the cancer is clear cell renal carcinoma (CCRCC).

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is a breast cancer.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is glioma.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is a prostate cancer or castration-resistant prostate cancer.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is a kidney cancer.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is a pancreatic cancer.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is an adenocarcinoma to the liver.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the cancer is a neovascular.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the cancer is renal cancer.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is a urothelial cancer.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is a brain cancer.

In some embodiments described herein, the subject has a tumor that expresses PSMA.

In some embodiments described herein, the subject has tumor-infiltrating T lymphocytes (TILs) in the tumor tissue.

“Increased number” refers to statistically significant increase in a subject when compared to a control. “Increased number” for example refers to statistically significant increase in the number of TILs in a subject (e.g. patient) pre- and post-treatment with a PSMA antibody or other therapeutic.

In some embodiments described herein, the subject has increased expression or activity of interferon-gamma (IFN-γ).

In some embodiments described herein the subject has been treated with an anti-PSMA antibody.

In some embodiments described herein, the subject is refractory to treatment with the anti-PSMA antibody.

Any of the PSMA or bispecific PSMA×CD3 antibodies of the invention described herein may be used in the methods of the invention.

The antibodies and fragments thereof as described herein may also be administered in combination therapy, i.e., combined with other therapeutic agents relevant for the disease or condition to be treated. Accordingly, in one embodiment, the antibody-containing medicament is for combination with one or more further therapeutic agent, such as a chemotherapeutic agent. In some embodiments, the other therapeutic agent is a radiopharmaceutical agent (such as radium-223 chloride), secondary hormone therapies (such as abiraterone or enzalutamide), and/or chemotherapies (docetaxel and cabazitaxel). Such combined administration may be simultaneous, separate or sequential, in any order. For simultaneous administration the agents may be administered as one composition or as separate compositions, as appropriate. a chemotherapeutic agent (e.g., docetaxel, carboplatin, fludarabine), abiraterone, hormonal therapy (e.g., flutamide, bicalutamide, nilutamide, cyproterone acetate, ketoconazole, aminoglutethimide, abarelix, degarelix, leuprolide, goserelin, triptorelin, buserelin, ARN-509), serine or tyrosine kinase inhibitor (e.g., PI3 kinase inhibitor SF1126) (e.g., lapatanib), multikinase inhibitor (e.g., sorafenib, sunitinib), VEGF inhibitor (e.g., bevacizumab), TAK-700, cancer vaccine (e.g., BPX-101, PEP223), Listeria-based vaccine, lenalidomide, TOK-001, IGF-1 receptor inhibitor (e.g., cixutumumab), TRC105, Aurora A kinase inhibitor (e.g., MLN8237), proteasome inhibitor (e.g., bortezomib), OGX-011, radioimmunotherapy (e.g., HuJ591-GS), HDAC inhibitor (e.g., valproic acid, SB939, LBH589), hydroxychloroquine, mTOR inhibitor (e.g., everolimus), dovitinib lactate, diindolylmethane, efavirenz, OGX-427, genistein, IMC-3G3, bafetinib, CP-675,206, radiation therapy, surgery, or a combination thereof.

In one embodiment, a method for treating a disorder involving cells expressing PSMA in a subject, which method comprises administration of a therapeutically effective amount of a bispecific antibody or fragment, such as a PSMA×CD3 bispecific antibody described herein, and radiotherapy to a subject in need thereof is provided. In one embodiment is provided a method for treating or preventing cancer, which method comprises administration of a therapeutically effective amount of a bispecific antibody or fragment, such as a PSMA x CD3 antibody described herein, and radiotherapy to a subject in need thereof. Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals to a patient is provided. The source of radiation may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used in practicing such methods include, e.g., radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-111.

Antibodies of the invention described herein may be administered in combination with a vaccine.

Exemplary vaccines are immunogenic agents, such as cancerous cells, purified tumor antigens (including recombinant proteins, antigen epitopes, peptides and carbohydrate molecules), tumor antigens delivered to a patient via gene therapy, cells, and cells transfected with genes encoding immune stimulating cytokines. Exemplary vaccines that may be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF, DNA-based vaccines, RNA-based vaccines, Listeria-based vaccines and viral transduction-based vaccines, peptides or prostate antigens (eg PSMA, STEAPI, PSCA), the cancer vaccine sipuleucel-T or peptides of lung cancer antigens. The cancer vaccine may be prophylactic or therapeutic.

Many experimental strategies for vaccination against tumors have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita, V. et al. (eds.), 1997, Cancer: Principles and Practice of Oncology. Fifth Edition). In one of these strategies, a vaccine is prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when the tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al., (1993) Proc Natl Acad Sci U.S.A. 90: 3539-43).

The antibodies of the invention described herein may be administered in combination with one or a collection of recombinant proteins and/or peptides expressed in or on a tumor in order to generate an immune response to these proteins. These proteins are normally viewed by the immune system as self-antigens and are therefore tolerant to them. The tumor antigen may also include the protein telomerase, which is required for the synthesis of telomeres of chromosomes and which is expressed in more than 85% of human cancers and in only a limited number of somatic tissues (Kim et al., (1994) Science 266: 2011-2013). Tumor antigens may also be “neo-antigens” expressed in or on cancer cells as a result of somatic mutations that alter protein sequence or create fusion proteins between two unrelated sequences (e.g., bcr-abl in the Philadelphia chromosome), or idiotype from B cell tumors. The tumor antigens may be antigen epitopes of prostate specific antigen (PSA), mesothelin, prostate-specific membrane antigen (PSMA), synovial sarcoma X2 (SSX2), NKX3.1, prostatic acidic phosphatase (PAP), or epidermal growth factor receptors, or peptides specific for variants of EGFR such as the well-known EGFRvIII overexpressed on tumor cells.

Other tumor vaccines may include the proteins from viruses implicated in human cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses (HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV), and Epstein-Barr virus (EBV). Another form of tumor specific antigens which may be used in combination with the antibodies of the invention described herein is purified heat shock proteins (HSP) isolated from the tumor tissue itself. HSP contain fragments of proteins from the tumor cells and are highly efficient at delivery to antigen presenting cells for eliciting tumor immunity (Suot and Srivastava (1995) Science 269:1585-1588; Tamura et al., (1997) Science 278:117-120).

Dendritic cells (DC) are potent antigen presenting cells that may be used to prime antigen-specific responses. DC's may be produced ex vivo and loaded with various protein and peptide antigens as well as tumor cell extracts (Nestle et al., (1998) Nature Medicine 4: 328-332). DCs may also be transduced by genetic means to express these tumor antigens. DCs have also been fused directly to tumor cells for the purposes of immunization (Kugler et al., (2000) Nature Medicine 6:332-336). As a method of vaccination, DC immunization may be effectively combined with the antibodies of the invention described herein to activate more potent anti-tumor responses.

In some embodiments described herein, the antibodies specifically binding PSMA of the invention or the bispecific PSMA×CD3 antibodies of the invention are administered in combination with a tumor vaccine comprising a peptide fragment of a prostate specific antigen, or a vector encoding the peptide fragment of a prostate specific antigen.

The antibodies of the invention described herein may be administered in combination with a standard of care cancer treatment.

The antibodies of the invention described herein may be administered in combination with a standard of care cancer chemotherapeutic regimes. In these instances, it may be possible to reduce the dose of chemotherapeutic reagent administered (Mokyr et al., (1998) Cancer Research 58: 5301-5304).

In some embodiments described herein, the antibodies of the invention may be administered in combination with one or more of other antibody molecules, chemotherapy, other anti-cancer therapy (e.g., targeted anti-cancer therapies, or oncolytic drugs), cytotoxic agents, cytokines, surgical and/or radiation procedures.

Exemplary cytotoxic agents that may be administered in combination with the antibodies of the invention described herein include hormone inhibitors, antimicrotubule agents, topoisomerase inhibitors, anti-metabolites, mitotic inhibitors, alkylating agents, anthracyclines, vinca alkaloids, intercalating agents, agents capable of interfering with a signal transduction pathway, agents that promote apoptosis, proteosome inhibitors, and radiation (e.g., local or whole body irradiation).

Standard of care therapeutics include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine (difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone@), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), vinorelbine (Navelbine®), Ibrutinib, idelalisib, and brentuximab vedotin.

Exemplary alkylating agents include, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes: uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil Nitrogen Mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune®), ifosfamide (Mitoxana®), melphalan (Alkeran®), Chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, Temozolomide (Temodar@), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar@), and Dacarbazine (DTIC-Dome®). Additional exemplary alkylating agents include, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® and Temodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®); Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®); Altretamine (also known as hexamethylmelamine (HMM), Hexalen®); Carmustine (BiCNU®) Bendamustine (Treanda®); Busulfan (Busulfex® and Myleran®); Carboplatin (Paraplatin®); Lomustine (also known as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® and Neosar®); Dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-Dome®); Altretamine (also known as hexamethylmelamine (HMM), Hexalen®); Ifosfamide (Ifex®); Prednumustine; Procarbazine (Matulane®); Mechlorethamine (also known as nitrogen mustard, mustine and mechloroethamine hydrochloride, Mustargen®); Streptozocin (Zanosar®); Thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex®); Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and Bendamustine HC1 (Treanda®).

Exemplary anthracyclines include, e.g., doxorubicin (Adriamycin® and Rubex®); bleomycin (Lenoxane®); daunorubicin (dauorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cerubidine®); daunorubicin liposomal (daunorubicin citrate liposome, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin; and desacetylravidomycin.

Exemplary vinca alkaloids that may be used in combination with the antibodies of the invention include vinorelbine tartrate (Navelbine®), Vincristine (Oncovin®), and Vindesine (Eldisine®); vinblastine (also known as vinblastine sulfate, vincaleukoblastine and VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®).

Exemplary proteosome inhibitors that may be used in combination with the antibodies of the invention, alone or in combination with another immunomodulator are bortezomib (Velcade®); carfilzomib (PX-171-007, (S)-4-Methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)-pentanamide); marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-Methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1 S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

In some embodiments described herein, the antibodies of the invention are administered in combination with a serine or tyrosine kinase inhibitor (e.g., a receptor tyrosine kinase (RTK) inhibitor). Exemplary tyrosine kinase inhibitor include an epidermal growth factor (EGF) pathway inhibitor (e.g., an epidermal growth factor receptor (EGFR) inhibitor), a vascular endothelial growth factor (VEGF) pathway inhibitor (e.g., a vascular endothelial growth factor receptor (VEGFR) inhibitor (e.g., a VEGFR-1 inhibitor, a VEGFR-2 inhibitor, a VEGFR-3 inhibitor), a platelet derived growth factor (PDGF) pathway inhibitor (e.g., a platelet derived growth factor receptor (PDGFR) inhibitor (e.g., a PDGFR-P3 inhibitor), a RAF-1 inhibitor, a KIT inhibitor and a RET inhibitor. In some embodiments, the second therapeutic is axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™, AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®, SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474), vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab (AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), alemtuzumab (CAMPATH®), gemtuzumab ozogamicin (MYLOTARG®), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, XL228, AEE788, AG-490, AST-6, BMS-599626, CUDC-101, PD153035, pelitinib (EKB-569), vandetanib (zactima), WZ3146, WZ4002, WZ8040, ABT-869 (linifanib), AEE788, AP24534 (ponatinib), AV-951 (tivozanib), axitinib, BAY 73-4506 (regorafenib), brivanib alaninate (BMS-582664), brivanib (BMS-540215), cediranib (AZD2171), CHIR-258 (dovitinib), CP 673451, CYC116, E7080, Ki8751, masitinib (AB1010), MGCD-265, motesanib diphosphate (AMG-706), MP-470, OSI-930, Pazopanib Hydrochloride, PD173074, Sorafenib Tosylate (Bay 43-9006), SU 5402, TSU-68 (SU6668), vatalanib, XL880 (GSK1363089, EXEL-2880). Selected tyrosine kinase inhibitors are chosen from sunitinib, erlotinib, gefitinib, or sorafenib. I some embodiments, the EGFR inhibitor os a bispecific EGFRc-Met antibody(EM-1 mAb) comprising the heavy and the light chains of SEQ DI NO:s 249, 250, 251 and 252 (US2014/0141000).

In some embodiments, the antibodies of the invention are administered in combination with Vascular Endothelial Growth Factor (VEGF) receptor inhibitors, including Bevacizumab (Avastin®), axitinib (Inlyta®); Brivanib alaninate (BMS-582664, (S)—((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate); Sorafenib (Nexavar@): Pazopanib (Votrient®); Sunitinib malate (Sutent®); Cediranib (AZD2171, CAS 288383-20-1); Vargatef (BIBF1120, CAS 928326-83-4); Foretinib (GSK1363089); Telatinib (BAY57-9352, CAS 332012-40-5); Apatinib (YN968D1, CAS 811803-05-1); Imatinib (Gleevec®); Ponatinib (AP24534, CAS 943319-70-8); Tivozanib (AV951, CAS 475108-18-0); Regorafenib (BAY73-4506, CAS 755037-03-7); Vatalanib dihydrochloride (PTK787, CAS 212141-51-0); Brivanib (BMS-540215, CAS 649735-46-6); Vandetanib (Caprelsa® or AZD6474); Motesanib diphosphate (AMG706, CAS 857876-30-3, N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide, described in PCT Publication No. WO 02/066470); Dovitinib dilactic acid (TKI258, CAS 852433-84-2); Linfanib (ABT869, CAS 796967-16-3); Cabozantinib (XL184, CAS 849217-68-1); Lestaurtinib (CAS 111358-88-4); N-[5-[[[5-(1,1-Dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide (BMS38703, CAS 345627-80-7); (3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); N-(3,4-Dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα, 5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine (XL647, CAS 781613-23-8); 4-Methyl-3-[[1-methyl-6-(3-pyridinyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]amino]-N-[3-(trifluoromethyl)phenyl]-benzamide (BHG712, CAS 940310-85-0); and Aflibercept (Eylea®).

Exemplary VEGF inhibitors include a monoclonal antibody that binds to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB 10709; a recombinant humanized anti-VEGF monoclonal antibody generated according to Presta et al., (1997) Cancer Res 57:4593-4599. In one embodiment, the anti-VEGF antibody is Bevacizumab (BV), also known as rhuMAb VEGF or AVASTIN®. It comprises mutated human IgG1 framework regions and antigen-binding complementarity-determining regions from the murine anti-hVEGF monoclonal antibody A.4.6.1 that blocks binding of human VEGF to its receptors. Bevacizumab and other humanized anti-VEGF antibodies are further described in U.S. Pat. No. 6,884,879. Additional anti-VEGF antibodies include the G6 or B20 series antibodies (e.g., G6-31, B20-4.1), as described in Int. Patent Publication Nos. WO2005/012359 and WO2005/044853, For additional antibodies see U.S. Pat. Nos. 7,060,269, 6,582,959, 6,703,020, 6,054,297, WO98/45332, WO 96/30046, WO94/10202, EP 0666868B1, U.S. Patent Application Publication Nos. US2006009360, US20050186208, US20030206899, US20030190317, US20030203409, and US20050112126; and Popkov et al., (2004) Journal of Immunological Methods 288: 149-164. Other antibodies include those that bind to a functional epitope on human VEGF comprising of residues F17, M18, D19, Y21, Y25, Q89, 191, K101, E103, and C104 or, alternatively, comprising residues F17, Y21, Q22, Y25, D63, 183 and Q89.

In some embodiments described herein, the antibodies of the invention are administered in combination with a PI3K inhibitor. In one embodiment, the PI3K inhibitor is an inhibitor of delta and gamma isoforms of PI3K. In another embodiment, the PI3K inhibitor is an inhibitor of beat isoforms of PI3K. Exemplary PI3K inhibitors that may be used are described in, e.g., WO 2010/036380, WO 2010/006086, WO 09/114870, WO 05/113556, GSK 2126458, GDC-0980, GDC-0941, Sanofi XL147, XL756, XL147, PF-46915032, BKM 120, CAL-101, CAL 263, SF1126, PX-886, and a dual PI3K inhibitor (e.g., Novartis BEZ235).

In some embodiments described herein, the antibodies of the invention are administered in combination with a mTOR inhibitor, e.g., one or more mTOR inhibitors chosen from one or more of rapamycin, temsirolimus (TORISEL®), AZD8055, BEZ235, BGT226, XL765, PF-4691502, GDC0980, SF1126, OSI-027, GSK1059615, KU-0063794, WYE-354, Palomid 529 (P529), PF-04691502, or PKI-587. ridaforolimus (formally known as deferolimus, (1R,2R,4S)-4-[(2R)-2 [(1R9S, 12S,15R,16E,18R,19,R21,R23 S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.04,9] hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03/064383); everolimus (Afinitor® or RAD001); rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3); emsirolimus, (5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-(SEQ ID NO: 237), inner salt (SF1126, CAS 936487-67-1), and XL765.

In some embodiments described herein, the antibodies of the invention are administered in combination with a BRAF inhibitor, e.g., GSK2118436, RG7204, PLX4032, GDC-0879, PLX4720, and sorafenib tosylate (Bay 43-9006).

In some embodiments described herein, the antibodies of the invention are administered in combination with an immunomodulatory agent. Targeting immune checkpoints such as programmed cell death protein 1 (PD1), programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory signals and enabling patients to produce an effective antitumour response. In some embodiments, the antibodies of the invention are administered in combination with an anti-PD1 (eg. nivolumab), anti-PDL (eg. MDX-1105) or anti-CTLA4 (eg. Ipilimumab). The ability of agonistic CD40 antibodies (referred to as αCD40) or CD40 ligand to stimulate immune responses and target tumors suggests such reagents have promise as cancer immunotherapeutics. In some embodiments, the antibodies of the invention are administered in combination with an anti-CD40 (eg. SGN-40, CP-870,893) or anti-CD40L (eg. BG9588).

In some embodiments described herein, the antibodies of the invention are administered in combination with a MEK inhibitor.

In some embodiments described herein, the antibodies of the invention administered in combination with the MEK inhibitor are used in the treatment of a prostate cancer, a melanoma, a colorectal cancer, a non-small cell lung cancer, an ovarian cancer, a breast cancer, a prostate cancer, a pancreatic cancer, a hematological malignancy or a renal cell carcinoma. In certain embodiments, the tumor tissue or cancer cell has a BRAF mutation (e.g., a BRAF V600E mutation), a BRAF wildtype, a KRAS wildtype or an activating KRAS mutation. The cancer may be at an early, intermediate or late stage. Any MEK inhibitor may be used in combination including, ARRY-142886, G02442104 (also known as GSK1120212), RDEA436, RDEA119/BAY 869766, AS703026, G00039805 (also known as AZD-6244 or selumetinib), BIX 02188, BIX 02189, CI-1040 (PD-184352), PD0325901, PD98059, U0126, GDC-0973 (Methanone, [3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]phenyl][3-hydroxy-3-(25)-2-piperidinyl-1-azetidinyl]-), G-38963, G02443714 (also known as AS703206), or a pharmaceutically acceptable salt or solvate thereof. Additional examples of MEK inhibitors are disclosed in WO 2013/019906, WO 03/077914, WO 2005/121142, WO 2007/04415, WO 2008/024725 and WO 2009/085983.

In some embodiments described herein, the antibodies of the invention are administered in combination with a JAK2 inhibitor, e.g., CEP-701, INCB18424, CP-690550 (tasocitinib).

In some embodiments described herein, the antibodies of the invention are administered in combination with paclitaxel or a paclitaxel agent, e.g., TAXOL®, protein-bound paclitaxel (e.g., ABRAXANE®). Exemplary paclitaxel agents include nanoparticle albumin-bound paclitaxel (ABRAXANE, marketed by Abraxis Bioscience), docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin, marketed by Protarga), polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX, marketed by Cell Therapeutic), the tumor-activated prodrug (TAP), ANGI05 (Angiopep-2 bound to three molecules of paclitaxel, marketed by ImmunoGen), paclitaxel-EC-1 (paclitaxel bound to the erbB2-recognizing peptide EC-1; see Li et al., Biopolymers (2007) 87:225-230), and glucose-conjugated paclitaxel (e.g., 2′-paclitaxel methyl 2-glucopyranosyl succinate, see Liu et al., (2007) Bioorganic & Medicinal Chemistry Letters 17:617-620).

In some embodiments described herein, the antibodies of the invention are administered in combination with a cellular immunotherapy (e.g., Provenge (e.g., Sipuleucel)), and optionally in combination with cyclophosphamide.

Exemplary therapeutic agents that may be used in combination with the antibodies of the invention for treatment of a prostate cancer include a chemotherapeutic agent (e.g., docetaxel, carboplatin, fludarabine), abiraterone, hormonal therapy (e.g., flutamide, bicalutamide, nilutamide, cyproterone acetate, ketoconazole, aminoglutethimide, abarelix, degarelix, leuprolide, goserelin, triptorelin, buserelin), tyrosine kinase inhibitor (e.g., dual kinase inhibitor (e.g., lapatanib), multikinase inhibitor (e.g., sorafenib, sunitinib), VEGF inhibitor (e.g., bevacizumab), TAK-700, cancer vaccine (e.g., BPX-101, PEP223), lenalidomide, TOK-001, IGF-1 receptor inhibitor (e.g., cixutumumab), TRC105, Aurora A kinase inhibitor (e.g., MLN8237), proteasome inhibitor (e.g., bortezomib), OGX-011, radioimmunotherapy (e.g., HuJ591-GS), HDAC inhibitor (e.g., valproic acid, SB939, LBH589), hydroxychloroquine, mTOR inhibitor (e.g., everolimus), dovitinib lactate, diindolylmethane, efavirenz, OGX-427, genistein, IMC-3G3, bafetinib, CP-675,206, ARN-509, radiation therapy, surgery, or a combination thereof.

Exemplary therapeutic agents that may be used in combination with the antibodies of the invention for treatment of pancreatic cancer include a chemotherapeutic agent, e.g., paclitaxel or a paclitaxel agent (e.g., a paclitaxel formulation such as TAXOL, an albumin-stabilized nanoparticle paclitaxel formulation (e.g., ABRAXANE) or a liposomal paclitaxel formulation); gemcitabine (e.g., gemcitabine alone or in combination with AXP107-11); other chemotherapeutic agents such as oxaliplatin, 5-fluorouracil, capecitabine, rubitecan, epirubicin hydrochloride, NC-6004, cisplatin, docetaxel (e.g., TAXOTERE), mitomycin C, ifosfamide; interferon; tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g., erlotinib, panitumumab, cetuximab, nimotuzumab); HER2/neu receptor inhibitor (e.g., trastuzumab); dual kinase inhibitor (e.g., bosutinib, saracatinib, lapatinib, vandetanib); multikinase inhibitor (e.g., sorafenib, sunitinib, XL184, pazopanib); VEGF inhibitor (e.g., bevacizumab, AV-951, brivanib); radioimmunotherapy (e.g., XR303); cancer vaccine (e.g., GVAX, survivin peptide); COX-2 inhibitor (e.g., celecoxib); IGF-1 receptor inhibitor (e.g., AMG 479, MK-0646); mTOR inhibitor (e.g., everolimus, temsirolimus); IL-6 inhibitor (e.g., CNTO 328); cyclin-dependent kinase inhibitor (e.g., P276-00, UCN-01); Altered Energy Metabolism-Directed (AEMD) compound (e.g., CPI-613); HDAC inhibitor (e.g., vorinostat); TRAIL receptor 2 (TR-2) agonist (e.g., conatumumab); MEK inhibitor (e.g., AS703026, selumetinib, GSK1120212); Raf/MEK dual kinase inhibitor (e.g., RO5126766); Notch signaling inhibitor (e.g., MK0752); monoclonal antibody-antibody fusion protein (e.g., L19IL2); curcumin; HSP90 inhibitor (e.g., tanespimycin, STA-9090); rIL-2; denileukin diftitox; topoisomerase 1 inhibitor (e.g., irinotecan, PEPO2); statin (e.g., simvastatin); Factor Vila inhibitor (e.g., PCI-27483); AKT inhibitor (e.g., RX-0201); hypoxia-activated prodrug (e.g., TH-302); metformin hydrochloride, gamma-secretase inhibitor (e.g., R04929097); ribonucleotide reductase inhibitor (e.g., 3-AP); immunotoxin (e.g., HuC242-DM4); PARP inhibitor (e.g., KU-0059436, veliparib); CTLA-4 inhbitor (e.g., CP-675,206, ipilimumab); AdV-tk therapy; proteasome inhibitor (e.g., bortezomib (Velcade), NPI-0052); thiazolidinedione (e.g., pioglitazone); NPC-1C; Aurora kinase inhibitor (e.g., R763/AS703569), CTGF inhibitor (e.g., FG-3019); siG12D LODER; and radiation therapy (e.g., tomotherapy, stereotactic radiation, proton therapy), surgery, and a combination thereof. In certain embodiments, a combination of paclitaxel or a paclitaxel agent, and gemcitabine can be used with the antibodies of the invention.

Exemplary therapeutic agents that may be used in combination with the antibodies of the invention for treatment of small cell lung cancer include a chemotherapeutic agent, e.g., etoposide, carboplatin, cisplatin, oxaliplatin, irinotecan, topotecan, gemcitabine, liposomal SN-38, bendamustine, temozolomide, belotecan, NK012, FR901228, flavopiridol); tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g., erlotinib, gefitinib, cetuximab, panitumumab); multikinase inhibitor (e.g., sorafenib, sunitinib); VEGF inhibitor (e.g., bevacizumab, vandetanib); cancer vaccine (e.g., GVAX); Bcl-2 inhibitor (e.g., oblimersen sodium, ABT-263); proteasome inhibitor (e.g., bortezomib (Velcade), NPI-0052), paclitaxel or a paclitaxel agent; docetaxel; IGF-1 receptor inhibitor (e.g., AMG 479); HGF/SF inhibitor (e.g., AMG 102, MK-0646); chloroquine; Aurora kinase inhibitor (e.g., MLN8237); radioimmunotherapy (e.g., TF2); HSP90 inhibitor (e.g., tanespimycin, STA-9090); mTOR inhibitor (e.g., everolimus); Ep-CAM/CD3-bispecific antibody (e.g., MTI10); CK-2 inhibitor (e.g., CX-4945); HDAC inhibitor (e.g., belinostat); SMO antagonist (e.g., BMS 833923); peptide cancer vaccine, and radiation therapy (e.g., intensity-modulated radiation therapy (IMRT), hypofractionated radiotherapy, hypoxia-guided radiotherapy), surgery, and combinations thereof.

Exemplary therapeutic agents that may be used in combination with the antibodies of the invention for treatment of non-small cell lung cancer include a chemotherapeutic agent, e.g., vinorelbine, cisplatin, docetaxel, pemetrexed disodium, etoposide, gemcitabine, carboplatin, liposomal SN-38, TLK286, temozolomide, topotecan, pemetrexed disodium, azacitidine, irinotecan, tegafur-gimeracil-oteracil potassium, sapacitabine); tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g., erlotinib, gefitinib, cetuximab, panitumumab, necitumumab, PF-00299804, nimotuzumab, RO5083945), MET inhibitor (e.g., PF-02341066, ARQ 197), PI3K kinase inhibitor (e.g., XL147, GDC-0941), Raf/MEK dual kinase inhibitor (e.g., RO5126766), PI3K/mTOR dual kinase inhibitor (e.g., XL765), SRC inhibitor (e.g., dasatinib), dual inhibitor (e.g., BIBW 2992, GSK1363089, ZD6474, AZD0530, AG-013736, lapatinib, MEHD7945A, linifanib), multikinase inhibitor (e.g., sorafenib, sunitinib, pazopanib, AMG 706, XL184, MGCD265, BMS-690514, R935788), VEGF inhibitor (e.g., endostar, endostatin, bevacizumab, cediranib, BIBF 1120, axitinib, tivozanib, AZD2171), cancer vaccine (e.g., BLP25 liposome vaccine, GVAX, recombinant DNA and adenovirus expressing L523S protein), Bcl-2 inhibitor (e.g., oblimersen sodium), proteasome inhibitor (e.g., bortezomib, carfilzomib, NPI-0052, MLN9708), paclitaxel or a paclitaxel agent, docetaxel, IGF-1 receptor inhibitor (e.g., cixutumumab, MK-0646, OSI 906, CP-751,871, BIIB022), hydroxychloroquine, HSP90 inhibitor (e.g., tanespimycin, STA-9090, AUY922, XL888), mTOR inhibitor (e.g., everolimus, temsirolimus, ridaforolimus), Ep-CAM/CD3-bispecific antibody (e.g., MT110), CK-2 inhibitor (e.g., CX-4945), HDAC inhibitor (e.g., MS 275, LBH589, vorinostat, valproic acid, FR901228), DHFR inhibitor (e.g., pralatrexate), retinoid (e.g., bexarotene, tretinoin), antibody-drug conjugate (e.g., SGN-15), bisphosphonate (e.g., zoledronic acid), cancer vaccine (e.g., belagenpumatucel-L), low molecular weight heparin (LMWH) (e.g., tinzaparin, enoxaparin), GSK1572932A, melatonin, talactoferrin, dimesna, topoisomerase inhibitor (e.g., amrubicin, etoposide, karenitecin), nelfinavir, cilengitide, ErbB3 inhibitor (e.g., MM-121, U3-1287), survivin inhibitor (e.g., YM155, LY2181308), eribulin mesylate, COX-2 inhibitor (e.g., celecoxib), pegfilgrastim, Polo-like kinase 1 inhibitor (e.g., BI 6727), TRAIL receptor 2 (TR-2) agonist (e.g., CS-1008), CNGRC peptide (SEQ ID NO: 225)-TNF alpha conjugate, dichloroacetate (DCA), HGF inhibitor (e.g., SCH 900105), SAR240550, PPAR-gamma agonist (e.g., CS-7017), gamma-secretase inhibitor (e.g., RO4929097), epigenetic therapy (e.g., 5-azacitidine), nitroglycerin, MEK inhibitor (e.g., AZD6244), cyclin-dependent kinase inhibitor (e.g., UCN-01), cholesterol-Fus1, antitubulin agent (e.g., E7389), farnesyl-OH-transferase inhibitor (e.g., lonafarnib), immunotoxin (e.g., BB-10901, SSI (dsFv) PE38), fondaparinux, vascular-disrupting agent (e.g., AVE8062), PD-L1 inhibitor (e.g., MDX-1105, MDX-1106), beta-glucan, NGR-hTNF, EMD 521873, MEK inhibitor (e.g., GSK1120212), epothilone analog (e.g., ixabepilone), kinesin-spindle inhibitor (e.g., 4SC-205), telomere targeting agent (e.g., KML-001), P70 pathway inhibitor (e.g., LY2584702), AKT inhibitor (e.g., MK-2206), angiogenesis inhibitor (e.g., lenalidomide), Notch signaling inhibitor (e.g., OMP-21M18), EGFR/c-Met bispecific antibody EM-1 as described in US2014/0141000A1, radiation therapy, surgery, and combinations thereof.

Exemplary therapeutic agents that may be used in combination with the antibodies of the invention for treatment of ovarian cancer include a chemotherapeutic agent (e.g., paclitaxel or a paclitaxel agent; docetaxel; carboplatin; gemcitabine; doxorubicin; topotecan; cisplatin; irinotecan, TLK286, ifosfamide, olaparib, oxaliplatin, melphalan, pemetrexed disodium, SJG-136, cyclophosphamide, etoposide, decitabine); ghrelin antagonist (e.g., AEZS-130), immunotherapy (e.g., APC8024, oregovomab, OPT-821), tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g., erlotinib), dual inhibitor (e.g., E7080), multikinase inhibitor (e.g., AZD0530, JI-101, sorafenib, sunitinib, pazopanib), ON 01910.Na), VEGF inhibitor (e.g., bevacizumab, BIBF 1120, cediranib, AZD2171), PDGFR inhibitor (e.g., IMC-3G3), paclitaxel, topoisomerase inhibitor (e.g., karenitecin, Irinotecan), HDAC inhibitor (e.g., valproate, vorinostat), folate receptor inhibitor (e.g., farletuzumab), angiopoietin inhibitor (e.g., AMG 386), epothilone analog (e.g., ixabepilone), proteasome inhibitor (e.g., carfilzomib), IGF-1 receptor inhibitor (e.g., OSI 906, AMG 479), PARP inhibitor (e.g., veliparib, AG014699, iniparib, MK-4827), Aurora kinase inhibitor (e.g., MLN8237, ENMD-2076), angiogenesis inhibitor (e.g., lenalidomide), DHFR inhibitor (e.g., pralatrexate), radioimmunotherapeutic agent (e.g., Hu3S193), statin (e.g., lovastatin), topoisomerase 1 inhibitor (e.g., NKTR-102), cancer vaccine (e.g., p53 synthetic long peptides vaccine, autologous OC-DC vaccine), mTOR inhibitor (e.g., temsirolimus, everolimus), BCR/ABL inhibitor (e.g., imatinib), ET-A receptor antagonist (e.g., ZD4054), TRAIL receptor 2 (TR-2) agonist (e.g., CS-1008), HGF/SF inhibitor (e.g., AMG 102), EGEN-001, Polo-like kinase 1 inhibitor (e.g., BI 6727), gamma-secretase inhibitor (e.g., RO4929097), Wee-1 inhibitor (e.g., MK-1775), antitubulin agent (e.g., vinorelbine, E7389), immunotoxin (e.g., denileukin diftitox), SB-485232, vascular-disrupting agent (e.g., AVE8062), integrin inhibitor (e.g., EMD 525797), kinesin-spindle inhibitor (e.g., 4SC-205), revlimid, HER2 inhibitor (e.g., MGAH22), ErrB3 inhibitor (e.g., MM-121), radiation therapy, and combinations thereof.

Exemplary therapeutics agents that may be used in combination with the antibodies of the invention for treatment of a renal cancer, e.g., a renal cell carcinoma (RCC) or metastatic RCC include an immune-based strategy (e.g., interleukin-2 or interferon-α), a targeted agent (e.g., a VEGF inhibitor such as a monoclonal antibody to VEGF, e.g., bevacizumab (Rini et al., (2010) J Clin Oncol 28(13):2137-2143)); a VEGF tyrosine kinase inhibitor such as sunitinib, sorafenib, axitinib and pazopanib (reviewed in Pal et al., (2014) Clin Advances in Hematology & Oncology 12(2):90-99); an RNAi inhibitor, or an inhibitor of a downstream mediator of VEGF signaling, e.g., an inhibitor of the mammalian target of rapamycin (mTOR), e.g., everolimus and temsirolimus (Hudes et al., (2007) N Engl J Med 356(22):2271-2281, Motzer et al., (2008) Lancet 372: 449-456).

PSMA-Specific Antibody Kits

Described herein are kits including the disclosed PSMA-specific antibodies or antigen-binding fragments thereof. The described kits may be used to carry out the methods of using the PSMA-specific antibodies or antigen-binding fragments provided herein, or other methods known to those skilled in the art. In some embodiments the described kits may include the antibodies or antigen-binding fragments described herein and reagents for use in detecting the presence of PSMA in a biological sample. Accordingly, the described kits may include one or more of the antibodies, or an antigen-binding fragment(s) thereof, described herein and a vessel for containing the antibody or fragment when not in use, instructions for use of the antibody or fragment, the antibody or fragment affixed to a solid support, and/or detectably labeled forms of the antibody or fragment, as described herein.

One embodiment of the invention is a kit comprising the antibody specifically binding PSMA of the invention.

Another embodiment of the invention is a kit comprising the bispecific PSMA×CD3 antibody comprising a first domain specifically binding PSMA and a second domain specifically binding CD3 of the invention.

The kit may be used for therapeutic uses and as diagnostic kits.

The kit may be used to detect the presence of PSMA, CD3 or PSMA and CD3 in a biological sample.

In some embodiments, the kit comprises the antibody of the invention described herein and reagents for detecting the antibody. The kit can include one or more other elements including: instructions for use; other reagents, e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, an antibody to a label or therapeutic agent, or a radioprotective composition; devices or other materials for preparing the antibody for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

In some embodiments, the kit comprises the antibody of the invention in a container and instructions for use of the kit.

In some embodiments, the antibody in the kit is labeled.

In some embodiments, the kit comprises the anti-PSMA antibody PSMB119, PSMB120, PSMB121, PSMB122, PSMB123, PSMB87, PSMB126, PSMB127, PSMB128, PSMB129, PSMB130, PSMB120, PSMB121, PSMB122, PSMB123, PSMB127, PSMB128, PSMB130, PSMB344, PSMB345, PSMB346, PSMB347, PSMB349, PSMB358, PSMB359, PSMB360, PSMB361, PSMB362, PSMB363, and PSMB365.

In some embodiments, the kit comprises the bispecific PSMA×CD3 antibody, PS3B22, PS3B23, PS3B25, PS3B27, PS3B28, or PS3B30.

Methods of Detecting PSMA or PSMA and CD3

One embodiment of the invention described herein is a method of detecting PSMA in a sample, comprising obtaining the sample, contacting the sample with the antagonistic antibody specifically binding PSMA of the invention, and detecting the antibody bound to PSMA in the sample.

One embodiment of the invention described herein is a method of detecting PSMA and CD3 in a sample, comprising obtaining the sample, contacting the sample with the bispecific antibody comprising a first domain specifically binding PSMA and a second domain specifically binding CD3 of the invention, and detecting the antibody bound to PSMA and CD3 in the sample.

In some embodiments described herein, the sample may be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, cells that are not tissue associated (i.e., free cells), tissues (e.g., surgically resected tumor tissue, biopsies, including fine needle aspiration), histological preparations, and the like.

The antibodies of the invention described herein bound to PSMA or PSMA and CD3 may be detected using known methods. Exemplary methods include direct labeling of the antibodies using fluorescent or chemiluminescent labels, or radiolabels, or attaching to the antibodies of the invention a moiety which is readily detectable, such as biotin, enzymes or epitope tags. Exemplary labels and moieties are ruthenium, 111In-DOTA, 111In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, poly-histidine (HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazin dyes, phenanthridine dyes, rhodamine dyes and Alexafluor® dyes.

The antibodies of the invention may be used in a variety of assays to detect PSMA or PSMA and CD3 in the sample. Exemplary assays are western blot analysis, radioimmunoassay, surface plasmon resonance, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS) or ELISA assay.

EMBODIMENTS

-   1) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, wherein the antibody or antigen binding     fragment (i) binds to cells expressing recombinant Pan troglodytes     PSMA, wherein the binding to cells is measured by flow cytometry     and (ii) binds recombinant Pan troglodytes PSMA extracellular domain     (SEQ ID NO:4) with an affinity of about 30 nM or less, wherein the     affinity is measured by Proteon XPR36 surface plasmon resonance     assay. -   2) The antibody of embodiment 1, wherein the antibody has one, two,     three or four of the following properties:     -   a) binds LNCaP cells with a calculated EC₅₀ of 20 nM or less and         binds Macaca fascicularis PSMA-expressing HEK cells with a         calculated EC₅₀ of 40 nM or less, wherein the difference in         calculated EC₅₀ between binding LNCaP cells and binding Macaca         fascicularis PSMA-expressing HEK cells is less than 5-fold, and         wherein the calculated EC₅₀ is measured in a whole cell binding         assay at 0° C. using flow cytometry,     -   b) binds recombinant PSMA ECD from human (SEQ ID NO: 7), Pan         troglodytes (SEQ ID NO:4) and Macaca fascicularis (SEQ ID NO: 5)         with an equilibrium dissociation constant (K_(D)) of 12 nM or         less, wherein the K_(D) is measured using Proteon surface         plasmon resonance assay ProteOn XPR36 system at +25° C.;     -   c) displays T-cell mediated killing of LNCaP cells, C42 cells,         human PSMA-expressing HEK cells or Macaca fascicularis         PSMA-expressing HEK cells when paired in a bispecific antibody         with anti-CD3 antibody CD3B219, wherein the T-cell mediated         killing is measured by Chromium-51 or by caspase 3/7 activation         assay or     -   d) recognizes a conformational epitope wherein the epitope is         comprised of residues 1138, F235, P237, G238, D244, Y299, Y300,         Q303, K304, E307, and K324-P326 of human PSMA (SEQ ID NO:3) -   3) The antibody of embodiment 2, comprising the HCDR1, the HCDR2,     the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 14, 15,     16, 17, 18 and 19, respectively. -   4) The antibody of embodiment 2, comprising the HCDR1, the HCDR2,     the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 20, 21,     22, 23, 12 and 24, respectively. -   5) The antibody of embodiment 2, comprising the HCDR1, the HCDR2,     the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 26,     27, 28, 29 and 30, respectively. -   6) The antibody of embodiment 2, comprising the HCDR1, the HCDR2,     the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37,     38, 39, 40 and 41, respectively. -   7) The antibody of embodiment 2, comprising the HCDR1, the HCDR2,     the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 42,     43, 11, 12 and 13, respectively. -   8) The antibody of embodiment 2, comprising the HCDR1, the HCDR2,     the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 44,     45, 46, 29 and 47, respectively. -   9) The antibody of embodiment 2, comprising the HCDR1, the HCDR2,     the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37,     48, 49, 50 and 51, respectively. -   10) The antibody of embodiment 2, comprising the HCDR1, the HCDR2,     the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37,     52, 49, 50 and 51, respectively. -   11) The antibody of embodiment 2, comprising the HCDR1, the HCDR2,     the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 8, 9,     10, 11, 12 and 13, respectively. -   12) The antibody of embodiment 2, comprising the HCDR1, the HCDR2,     the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 32,     33, 34, 12 and 35, respectively. -   13) The antibody of embodiment 2, comprising the HCDR1, the HCDR2,     the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 53, 54,     55, 23, 12 and 35, respectively. -   14) The antibody of embodiment 2, comprising the HCDR1, the HCDR2,     the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 122,     123, 124, 23, 12 and 24, respectively. -   15) The antibody of embodiment 2, comprising a heavy chain variable     region (VH) of SEQ ID NO: 60, 62, 64, 66, 68, 70, 72, 74, 75, 77,     79, or 160. -   16) The antibody of embodiment 15, comprising a light chain variable     region (VL) of SEQ ID NOs: 61, 63, 65, 67, 69, 71, 73, 61, 76, or     78. -   17) The antibody of embodiment 15, comprising the VH of SEQ ID NO:     62 and the VL of SEQ ID NO: 63. -   18) The antibody of embodiment 15, comprising the VH of SEQ ID NO:     64 and the VL of SEQ ID NO: 65. -   19) The antibody of embodiment 15, comprising the VH of SEQ ID NO:     66 and the VL of SEQ ID NO: 67. -   20) The antibody of embodiment 15, comprising the VH of SEQ ID NO:     72 and the VL of SEQ ID NO: 73. -   21) The antibody of embodiment 15, comprising the VH of SEQ ID NO:     74 and the VL of SEQ ID NO: 61. -   22) The antibody of embodiment 15, comprising the VH of SEQ ID NO:     75 and the VL of SEQ ID NO: 76. -   23) The antibody of embodiment 15, comprising the VH of SEQ ID NO:     77 and the VL of SEQ ID NO: 78. -   24) The antibody of embodiment 15, comprising the VH of SEQ ID NO:     79 and the VL of SEQ ID NO: 78. -   25) The antibody of embodiment 15, comprising the VH of SEQ ID NO:     160 and the VL of SEQ ID NO: 65. -   26) The antibody of embodiment 15, comprising the VH of SEQ ID NO:     60 and the VL of SEQ ID NO: 61. -   27) The antibody of embodiment 15, comprising the VH of SEQ ID NO:     68 and the VL of SEQ ID NO: 69.     -   The antibody of embodiment 15, comprising the VH of SEQ ID NO:         70 and the VL of SEQ ID NO: 71. -   28) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 42, 43, 11, 12 and     13, respectively. -   29) The antibody of embodiment 28, wherein the antibody comprises     the VH of SEQ ID NO: 74 and the VL of SEQ ID NO: 61. -   30) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 14, 15, 16, 17, 18 and     19, respectively. -   31) The antibody of embodiment 30, wherein the antibody comprises     the VH of SEQ ID NO: 62 and the VL of SEQ ID NO: 63. -   32) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29 and     30, respectively. -   33) The antibody of embodiment 32, wherein the antibody comprises     the VH of SEQ ID NO: 66 and the VL of SEQ ID NO: 67. -   34) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 38, 39, 40 and     41, respectively -   35) The antibody of embodiment 34, wherein the antibody comprises     the VH of SEQ ID NO: 72 and the VL of SEQ ID NO: 73. -   36) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 122, 123, 124, 23, 12,     and 24, respectively. -   37) The antibody of embodiment 36, wherein the antibody comprises     the VH of SEQ ID NO: 160 and the VL of SEQ ID NO: 65. -   38) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 8, 9, 10, 11, 12, and     13, respectively. -   39) The antibody of embodiment 38, wherein the antibody comprises     the VH of SEQ ID NO:60 and the VL of SEQ ID NO:61. -   40) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 20, 21, 22, 23, 12,     and 24, respectively. -   41) The antibody of embodiment 40, wherein the antibody comprises     the VH of SEQ ID NO:64 and the VL of SEQ ID NO:65. -   42) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 32, 33, 34, 12,     and 35, respectively. -   43) The antibody of embodiment 42, wherein the antibody comprises     the VH of SEQ ID NO:70 and the VL of SEQ ID NO:71. -   44) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 44, 45, 46, 29,     and 47, respectively. -   45) The antibody of embodiment 44, wherein the antibody comprises     the VH of SEQ ID NO:75 and the VL of SEQ ID NO:76. -   46) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 48, 49, 50,     and 51, respectively. -   47) The antibody of embodiment 46, wherein the antibody comprises     the VH of SEQ ID NO:77 and the VL of SEQ ID NO:78. -   48) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 52, 49, 50,     and 51, respectively. -   49) The antibody of embodiment 48, wherein the antibody comprises     the VH of SEQ ID NO:79 and the VL of SEQ ID NO:78. -   50) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 53, 54, 55, 23, 12,     and 35, respectively. -   51) The antibody of embodiment 50, wherein the antibody comprises     the VH of SEQ ID NO:68 and the VL of SEQ ID NO:69. -   52) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 29,     and 30, respectively. -   53) The antibody of embodiment 52, wherein the antibody comprises     the VH of SEQ ID NO:138 and the VL of SEQ ID NO:67. -   54) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 131, 29,     and 132, respectively. -   55) The antibody of embodiment 54, wherein the antibody comprises     the VH of SEQ ID NO: 138 and the VL of SEQ ID NO: 142. -   56) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 133,     and 132, respectively. -   57) The antibody of embodiment 56, wherein the antibody comprises     the VH of SEQ ID NO: 138 and the VL of SEQ ID NO:143. -   58) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29,     and 30, respectively. -   59) The antibody of embodiment 58, wherein the antibody comprises     the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 167. -   60) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29,     and 136, respectively. -   61) The antibody of embodiment 60, wherein the antibody comprises     the VH of SEQ ID NO: 140 and the VL of SEQ ID NO:144. -   62) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29,     and 30, respectively. -   63) The antibody of embodiment 62, wherein the antibody comprises     the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 167. -   64) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 131, 29,     and 132, respectively. -   65) The antibody of embodiment 64, wherein the antibody comprises     the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 142. -   66) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 133,     and 132, respectively. -   67) The antibody of embodiment 66, wherein the antibody comprises     the VH of SEQ ID NO: 140 and the VL of SEQ ID NO:143. -   68) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29,     and 136, respectively. -   69) The antibody of embodiment 68, wherein the antibody comprises     the VH of SEQ ID NO: 139 and the VL of SEQ ID NO:144. -   70) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 131, 29,     and 132, respectively. -   71) The antibody of embodiment 70, wherein the antibody comprises     the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 142. -   72) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 133,     and 132, respectively. -   73) The antibody of embodiment 72, wherein the antibody comprises     the VH of SEQ ID NO: 139 and the VL of SEQ ID NO:143. -   74) An isolated recombinant anti-PSMA antibody or antigen-binding     fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the     LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 137, 27, 28, 133,     and 132, respectively. -   75) The antibody of embodiment 74, wherein the antibody comprises     the VH of SEQ ID NO: 141 and the VL of SEQ ID NO: 143. -   76) The antibody of any one of embodiments 1-75, wherein the     antibody is human or humanized. -   77) The antibody of embodiment 76, wherein the antibody is of IgG4     or IgG1 isotype. -   78) The antibody of embodiment 77, comprising one, two, three, four,     five, six, seven, eight, nine or ten substitutions in the antibody     Fc. -   79) The antibody of embodiment 77, comprising     -   a) L234A, L235A, G237A, P238S, H268A, A330S and P331S         substitutions;     -   b) V234A, G237A, P238S, H268A, V309L, A330S and P331S         substitutions;     -   c) F234A, L235A, G237A, P238S and Q268A substitutions,     -   d) L234A, L235A or L234A and L235A substitutions;     -   e) F234A, L235A or F234A and L235A substitutions; or     -   f) V234A substitution, wherein residue numbering is according to         the EU Index. -   80) The antibody of embodiment 79, comprising S228P, F234A and L235A     substitutions, wherein residue numbering is according to the EU     Index. -   81) The antibody of any one of embodiments 1-80, wherein the     antibody is bispecific. -   82) The antibody of embodiment 76, wherein the antibody specifically     binds PSMA and specifically binds CD3, CD5, CD28, CD16, CD16A, CD25,     CD38, CD44, CD56, CD69, CD94, CD335 (NKp46), CD336, (NKp44), CD337     (NKp30), NKp80, NKG2C and NKG2D, DNAM, NCRs, CD18, CD89, CD18, CD32,     CD64, CD64 and CD35. -   83) A pharmaceutical composition comprising the antibody of any one     of embodiments 1-82 and a pharmaceutically accepted carrier. -   84) A polynucleotide encoding the antibody VH of embodiment 15, the     antibody VL of embodiment 16, or the antibody VH and the antibody VL     of embodiment 15 and 16. -   85) A polynucleotide encoding the antibody VH, the antibody VL, or     the antibody VH and the antibody VL of any one of embodiments 28-75. -   86) A vector comprising the polynucleotide of embodiment 84. -   87) A vector comprising the polynucleotide of embodiment 85. -   88) A host cell comprising the vector of embodiment 86. -   89) A host cell comprising the vector of embodiment 87. -   90) A method of producing the antibody of embodiment 1, comprising     culturing the host cell of embodiment 89 in conditions that the     antibody is expressed, and recovering the antibody produced by the     host cell. -   91) A method of treating a cancer in a subject, comprising     administering a therapeutically effective amount of the isolated     antibody of any one of embodiments 1-82 to the subject in need     thereof for a time sufficient to treat the cancer. -   92) The method of embodiment 91, wherein the cancer is a solid     tumor, malignancy or a tumor neovasculature. -   93) The method of embodiment 92, wherein the solid tumor is a     prostate cancer or a colorectal cancer, a gastric cancer, a clear     cell renal carcinoma, a bladder cancer, a lung cancer, a squamous     cell carcinoma, a glioma, a breast cancer, a kidney cancer, a     neovascular disorder, a clear cell renal carcinoma (CCRCC), a     pancreatic cancer, a renal cancer, a urothelial cancer and an     adenocarcinaoma to the liver. -   94) The method of embodiment 93, wherein the prostate cancer is a     refractory prostate cancer, a prostatic intraepithelial neoplasia,     an androgen independent prostate cancer, a malignant prostate     cancer. -   95) The method of any one of embodiments 90-94, wherein the antibody     is administered in combination with a second therapeutic agent. -   96) The method of embodiment 95, wherein the second therapeutic     agent is a standard of care drug for treatment of the solid tumor or     malignancy or a tumor neovasculature. -   97) The method of embodiment 96, wherein the second therapeutic     agent is an hormone inhibitor, an antimicrotubule agent, a kinase     inhibitor, an immunomodulating agent, a topoisomerase inhibitor, an     anti-metabolite, a mitotic inhibitor, an alkylating agent, an     anthracycline, a vinca alkaloid, an intercalating agent, an agent     capable of interfering with a signal transduction pathway, an agent     that promotes apoptosis, a proteosome inhibitor or radiation. -   98) The method of embodiment 96, wherein the second therapeutic     agent is a vaccine. -   99) The method of embodiment 98, wherein the vaccine is a     polypeptide or fragment thereof, or a DNA or a RNA encoding the     polypeptide or fragment thereof expressed on tumor cells. -   100) The method of embodiment 99, wherein the polypeptide is PSMA,     mesothelin, EGFR or EGFRvIII. -   101) The method of embodiment 95, wherein the second therapeutic     agent is administered simultaneously, sequentially or separately. -   102) The method of any one of embodiments 91-101, wherein the     subject is treated or is being treated with radiation therapy. -   103) The method of any one of embodiments 91-101 wherein the subject     has had or will undergo surgery. -   104) The method of any one of embodiments 46-58, wherein the     isolated antibody comprises the VH of SEQ ID NO: 66 and the VL of     SEQ ID NO: 67. -   105) The antibody of any one of embodiments 1-82 for use in therapy. -   106) An anti-idiotypic antibody binding to the antibody of any one     of embodiments 1-82. -   107) A bispecific antibody comprising a first domain that     specifically binds PSMA and a second domain that specifically binds     CD3, wherein the first domain comprises:     -   a) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 31, 42, 43, 11, 12 and 13, respectively;     -   b) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 14, 15, 16, 17, 18 and 19, respectively;     -   c) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively;     -   d) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 36, 37, 38, 39, 40 and 41, respectively;     -   e) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 122, 123, 124, 23, 12, and 24,         respectively;     -   f) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 8, 9, 10, 11, 12, and 13, respectively;     -   g) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 14, 15, 16, 17, 18, and 19, respectively;     -   h) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 20, 21, 22, 23, 12, and 24, respectively;     -   i) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 31, 32, 33, 34, 12, and 35, respectively;     -   j) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 31, 44, 45, 46, 29, and 47, respectively;     -   k) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 36, 37, 48, 49, 50, and 51, respectively;     -   l) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 53, 54, 55, 23, 12, and 35, respectively;     -   m) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 29, and 30, respectively;     -   n) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 130, 27, 131, 29, and 132,         respectively;     -   o) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 133, and 132,         respectively;     -   p) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29, and 30, respectively;     -   q) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29, and 136, respectively;     -   r) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29, and 30, respectively;     -   s) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 135, 27, 131, 29, and 132,         respectively;     -   t) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 133, and 132,         respectively;     -   u) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29, and 136, respectively;     -   v) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 134, 27, 131, 29, and 132,         respectively;     -   w) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 133, and 132,         respectively; or     -   x) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 137, 27, 28, 133, and 132,         respectively. -   108) The bispecific antibody of embodiment 107, wherein the first     domain comprises:     -   a) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 31, 42, 43, 11, 12 and 13, respectively,         and the VH of SEQ ID NO: 74 and the VL of SEQ ID NO: 61;     -   b) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 14, 15, 16, 17, 18 and 19, respectively,         and the VH of SEQ ID NO: 62 and the VL of SEQ ID NO: 63;     -   c) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively,         and the VH of SEQ ID NO: 66 and the VL of SEQ ID NO: 67;     -   d) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 36, 37, 38, 39, 40 and 41, respectively,         and the VH of SEQ ID NO: 72 and the VL of SEQ ID NO: 73;     -   e) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 122, 123, 124, 23, 12, and 24,         respectively, and the VH of SEQ ID NO: 160 and the VL of SEQ ID         NO: 65;     -   f) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 8, 9, 10, 11, 12, and 13, respectively, and         the VH of SEQ ID NO:60 and the VL of SEQ ID NO:61;     -   g) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 14, 15, 16, 17, 18, and 19, respectively,         and the VH of SEQ ID NO:62 and the VL of SEQ ID NO:63;     -   h) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 20, 21, 22, 23, 12, and 24, respectively,         and VH of SEQ ID NO:64 and the VL of SEQ ID NO:65;     -   i) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 31, 32, 33, 34, 12, and 35, respectively,         and the VH of SEQ ID NO:70 and the VL of SEQ ID NO:71;     -   j) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 31, 44, 45, 46, 29, and 47, respectively,         the VH of SEQ ID NO:75 and the VL of SEQ ID NO:76;     -   k) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 36, 37, 48, 49, 50, and 51, respectively,         and the VH of SEQ ID NO:77 and the VL of SEQ ID NO:78;     -   l) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 53, 54, 55, 23, 12, and 35, respectively,         and the VH of SEQ ID NO:68 and the VL of SEQ ID NO:69;     -   m) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 29, and 30, respectively,         and the VH of SEQ ID NO:138 and the VL of SEQ ID NO:67;     -   n) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 130, 27, 131, 29, and 132,         respectively, and the VH of SEQ ID NO:138 and the VL of SEQ ID         NO: 142;     -   o) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 133, and 132,         respectively, and the VH of SEQ ID NO:138 and the VL of SEQ ID         NO:143;     -   p) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29, and 30, respectively,         and the VH of SEQ ID NO:139 and the VL of SEQ ID NO:167;     -   q) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29, and 136, respectively,         and the VH of SEQ ID NO:140 and the VL of SEQ ID NO:144;     -   r) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29, and 30, respectively,         and the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 167;     -   s) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 135, 27, 131, 29, and 132, respectively         and the VH of SEQ ID NO:140 and the VL of SEQ ID NO: 142;     -   t) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 133, and 132,         respectively, and the VH of SEQ ID NO:140 and the VL of SEQ ID         NO:143;     -   u) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29, and 136, respectively;         and the VH of SEQ ID NO:139 and the VL of SEQ ID NO: 144;     -   v) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 134, 27, 131, 29, and 132,         respectively, and the VH of SEQ ID NO:139 and the VL of SEQ ID         NO: 142;     -   w) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 133, and 132,         respectively, and the VH of SEQ ID NO:139 and the VL of SEQ ID         NO:143; or     -   x) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the         LCDR3 of SEQ ID NOs: 25, 137, 27, 28, 133, and 132, respectively         and the VH of SEQ ID NO:141 and the VL of SEQ ID NO: 143. -   109) An isolated bispecific PSMA/CD3 antibody comprising a first     domain that (i) binds to cells expressing recombinant Pan     troglodytes PSMA, wherein the binding to cells is measured by flow     cytometry and (ii) binds recombinant Pan troglodytes PSMA     extracellular domain (SEQ ID NO:4) with an affinity of about 30 nM     or less, wherein the affinity is measured by Proteon surface plasmon     resonance assay specifically binding PSMA, and a second domain     specifically binding CD3. -   110) The bispecific PSMA×CD3 antibody of embodiment 109, wherein the     antibody     -   a) binds LNCaP cells with a calculated EC₅₀ of 20 nM or less and         binds Macaca fascicularis PSMA-expressing HEK cells with a         calculated EC₅₀ of 40 nM or less, wherein the difference in         calculated EC₅₀ between binding LNCaP cells and binding Macaca         fascicularis PSMA-expressing HEK cells is less than 5-fold, and         wherein the calculated EC₅₀ is measured in a whole cell binding         assay at 0° C. using flow cytometry,     -   b) binds recombinant PSMA ECD from human(SEQ ID NO:7), Pan         troglodytes (SEQ ID NO:4) and Macaca fascicularis (SEQ ID NO:5)         with an equilibrium dissociation constant (K_(D)) of 12 nM or         less, wherein the K_(D) is measured using Proteon surface         plasmon resonance assay ProteOn XPR36 system at +25° C.;     -   c) displays T-cell mediated killing of LNCaP cells, C42 cells,         human PSMA-expressing HEK cells or Macaca fascicularis         PSMA-expressing HEK cells, wherein the T-cell mediated killing         is measured by Chromium-51 or by caspase 3/7 activation assay or     -   d) recognizes a conformational epitope wherein the epitope is         comprised of residues I1138, F235, P237, G238, D244, Y299, Y300,         Q303, K304, E307, and K324-P326 of human PSMA (SEQ ID NO:3). -   111) The bispecific PSMA×CD3 antibody of embodiment 109, wherein the     antibody binds to T-cells. -   112) The bispecific PSMA×CD3 antibody of embodiment 109, wherein the     first domain comprises     -   a) the heavy chain complementarity determining region 1 (HCDR1),         a HCDR2 and a HCDR3 of SEQ ID NOs: 14, 15 and 16, respectively;         and the light chain complementarity determining region 1         (LCDR1), a LCDR2 and a LCDR3 of SEQ ID NOs: 17, 18 and 19,         respectively;     -   b) the HCDR1, the HCDR2 and the HCDR3 of SEQ ID NOs: 20, 21 and         22, respectively, and the LCDR1, the LCDR2 and the LCDR3 of SEQ         ID NOs: 23, 12 and 24, respectively;     -   c) the HCDR1, the HCDR2 and the HCDR3 of SEQ ID NOs: 25, 26 and         27, respectively, and the LCDR1, the LCDR2 and the LCDR3 of SEQ         ID NOs: 28, 29 and 30, respectively,     -   d) the HCDR1, the HCDR2 and the HCDR3 of SEQ ID NOs: 31, 44 and         45, respectively, and the LCDR1, the LCDR2 and the LCDR3 of SEQ         ID NOs: 46, 29 and 47, respectively;     -   e) the HCDR1, the HCDR2 and the HCDR3 of SEQ ID NOs: 31, 42 and         43, respectively, and the LCDR1, the LCDR2 and the LCDR3 of SEQ         ID NOs: 11, 12 and 13, respectively; or     -   f) the HCDR1, the HCDR2 and the HCDR3 of SEQ ID NOs: 122, 123         and 124, respectively, and the LCDR1, the LCDR2 and the LCDR3 of         SEQ ID NOs: 23, 12 and 24, respectively. -   113) The bispecific PSMA×CD3 antibody of embodiment 109, wherein the     first domain comprises the HCDR1, the HCDR2 and the HCDR3 of     -   a) SEQ ID NOs: 14, 15 and 16, respectively;     -   b) SEQ ID NOs: 20, 21 and 22, respectively;     -   c) SEQ ID NOs: 25, 26 and 27, respectively;     -   d) SEQ ID NOs: 31, 44 and 45, respectively;     -   e) SEQ ID NOs: 31, 42 and 43, respectively; or     -   f) SEQ ID NOs: 122, 123 and 124, respectively. -   114) The bispecific PSMA×CD3 antibody of embodiment 109, wherein the     first domain comprises the LCDR1, the LCDR2 and the LCDR3 of     -   a) SEQ ID NOs: 17, 18 and 19, respectively;     -   b) SEQ ID NOs: 23, 12 and 24, respectively;     -   c) SEQ ID NOs: 28, 29 and 30, respectively;     -   d) SEQ ID NOs: 46, 29 and 47, respectively;     -   e) SEQ ID NOs: 11, 12 and 13, respectively; or     -   f) SEQ ID NOs: 23, 12 and 24, respectively. -   115) The bispecific PSMA×CD3 antibody of embodiment 109, wherein     -   a) the first domain comprises a heavy chain variable region (VH)         of SEQ ID NO: 62 and a light chain variable region (VL) of SEQ         ID NO: 63, and the second domain comprises the VH of SEQ ID NO:         104 and the VL of SEQ ID NO: 105     -   b) the first domain comprises the VH of SEQ ID NO: 64 and the VL         of SEQ ID NO: 65, and the second domain comprises the VH of SEQ         ID NO: 104 and the VL of SEQ ID NO: 105;     -   c) the first domain comprises the VH of SEQ ID NO: 66 and the VL         of SEQ ID NO: 67, and the second domain comprises the VH of SEQ         ID NO: 104 and the VL of SEQ ID NO: 105;     -   d) the first domain comprises the VH of SEQ ID NO: 75 and the VL         of SEQ ID NO: 76, and the second domain comprises the VH of SEQ         ID NO: 104 and the VL of SEQ ID NO: 105;     -   e) the first domain comprises the VH of SEQ ID NO: 74 and the VL         of SEQ ID NO: 61, and the second domain comprises the VH of SEQ         ID NO: 104 and the VL of SEQ ID NO: 105;     -   f) the first domain comprises the VH of SEQ ID NO: 160 and the         VL of SEQ ID NO: 65, and the second domain comprises the VH of         SEQ ID NO: 104 and the VL of SEQ ID NO: 105. -   116) The bispecific PSMA×CD3 antibody of embodiment 109, comprising     a first heavy chain (HC1), a first light chain (LC1), a second heavy     chain (HC2) and a second light chain (LC2), wherein the HC1 and the     LC1 comprise the amino acid sequences of     -   a) SEQ ID NOs: 84 and 85, respectively;     -   b) SEQ ID NOs: 86 and 87, respectively;     -   c) SEQ ID NOs: 88 and 89, respectively;     -   d) SEQ ID NOs: 125 and 91, respectively;     -   e) SEQ ID NOs: 94 and 95, respectively; or     -   f) SEQ ID NOs: 96 and 83, respectively. -   117) The bispecific PSMA×CD3 antibody of embodiment 116, wherein the     HC2 and the LC2 comprises SEQ ID NOs: 110 and 111, respectively. -   118) The bispecific PSMA×CD3 antibody of embodiment 109, comprising     the HC1, the LC1, the HC2 and the LC2 of     -   a) SEQ ID NOs: 84, 85, 110 and 111, respectively;     -   b) SEQ ID NOs: 86, 87, 110 and 111, respectively;     -   c) SEQ ID NOs: 88, 89, 110, 111, respectively;     -   d) SEQ ID NOs: 125, 91, 110 and 111, respectively;     -   e) SEQ ID NOs: 94, 95, 110 and 111, respectively;     -   f) SEQ ID NOs: 96, 83, 110 and 111, respectively. -   119) The bispecific PSMA×CD3 antibody of any one of embodiments     109-118, wherein the antibody is human or humanized. -   120) The bispecific PSMA×CD3 antibody of embodiment 119, wherein the     antibody is of IgG1, IgG2, IgG3 or IgG4 isotype. -   121) The bispecific PSMA×CD3 antibody of embodiment 120, wherein the     antibody is of IgG1 or IgG4 isotype. -   122) The bispecific PSMA×CD3 antibody of embodiment 120 or 121,     having one, two, three, four, five, six, seven, eight, nine or ten     substitutions in an antibody Fc. -   123) The bispecific PSMA×CD3 antibody of embodiment 121, comprising:     -   a) L234A, L235A, G237A, P238S, H268A, A330S and P331S         substitutions;     -   b) V234A, G237A, P238S, H268A, V309L, A330S and P331 S         substitutions;     -   c) F234A, L235A, G237A, P238S and Q268A substitutions;     -   d) L234A, L235A or L234A and L235A substitutions;     -   e) F234A, L235A or F234A and L235A substitutions;     -   f) V234A substitution; or     -   g) S228P, F234A and L235A substitutions, wherein residue         numbering is according to the EU Index. -   124) The bispecific PSMA×CD3 antibody of any one of embodiments     109-123, comprising at least one substitution in an antibody CH3     constant domain. -   125) The bispecific PSMA×CD3 antibody of embodiment 124, wherein the     substitution in the antibody CH3 constant domain is 409R, F405L or     F405L/R409K substitution, wherein residue numbering is according to     the EU Index. -   126) The bispecific PSMA×CD3 antibody of embodiment 124, wherein the     antibody comprises     -   a) F405L substitution in the HC1 and 409R substitution in the         HC2, wherein the antibody is of IgG1 isotype; b) V234A, G237A,         P238S, H268A, V309L, A330S, P331S and F405L substitutions in the         HC1 and V234A, G237A, P238S, H268A, V309L, A330S, P331S and 409R         substitutions in the HC2, wherein the antibody is of IgG1         isotype; or     -   b) S228P substitution in the HC1 and S228P, F405L and R409K         substitution in the HC2, wherein the antibody is of IgG4         isotype. -   127) A pharmaceutical composition comprising the bispecific PSMA×CD3     antibody of any one of embodiments 109-126 and a pharmaceutically     accepted carrier. -   128) A polynucleotide encoding the bispecific PSMA×CD3 antibody HC1,     LC1, HC2 or LC2 of embodiment 118. -   129) A vector comprising the polynucleotide encoding the HC1, the     LC1, the HC2, the LC2, the HC1 and the LC1 or the HC2 and the LC2 of     embodiment 128. -   130) An isolated host cell comprising the vector of embodiment 129. -   131) A method of producing the bispecific PSMA×CD3 antibody of     embodiment 118, comprising culturing the host cell of embodiment 130     in conditions that the antibody is expressed, and recovering and     purifying the bispecific PSMA×CD3 antibody produced by the host     cell. -   132) A method of producing the bispecific PSMA×CD3 antibody of     embodiment 118, comprising:     -   a) combining a monospecific bivalent PSMA antibody having two         identical HC1 and two identical LC1 and a monospecific bivalent         CD3 antibody having two identical HC2 and two identical LC2 in a         mixture of about 1:1 molar ratio;     -   b) introducing a reducing agent into the mixture;     -   c) incubating the mixture about ninety minutes to about six         hours;     -   d) removing the reducing agent; and     -   e) purifying the bispecific PSMA×CD3 antibody that comprises the         HC1, the LC1, the HC2 and the LC2. -   133) The method of embodiment 132, wherein the reducing agent is     2-mercaptoethanolamine (2-MEA). -   134) The method of embodiment 133, wherein     -   h) the 2-MEA is present at a concentration of about 25 mM to         about 75 mM; and     -   i) the incubating step is performed at a temperature of about         25° C. to about 37° C. -   135) A method of treating a cancer in a subject, comprising     administering a therapeutically effective amount of the isolated     PSMA×CD3 bispecific antibody of any one of embodiments 109-126 to     the subject in need thereof for a time sufficient to treat the     cancer. -   136) The method of embodiment 135, wherein the cancer is a solid     tumor, malignancy or a tumor neovasculature. -   137) The method of embodiment 136, wherein the solid tumor is a     prostate cancer or a colorectal cancer, a gastric cancer, a clear     cell renal carcinoma, a bladder cancer, a lung cancer, a squamous     cell carcinoma, a glioma, a breast cancer, a kidney cancer, a     neovascular disorder, a clear cell renal carcinoma (CCRCC), a     pancreatic cancer, a renal cancer, a urothelial cancer and an     adenocarcinaoma to the liver. -   138) The method of embodiment 137, wherein the prostate cancer is a     refractory prostate cancer, a prostatic intraepithelial neoplasia,     an androgen independent prostate cancer, a malignant prostate     cancer. -   139) The method of any one of embodiments 135-138, wherein the     antibody is administered in combination with a second therapeutic     agent. -   140) The method of embodiment 139, wherein the second therapeutic     agent is a standard of care drug for treatment of the solid tumor or     malignancy or a tumor neovasculature. -   141) The method of embodiment 139, wherein the second therapeutic     agent is a hormone inhibitor, an antimicrotubule agent, a     topoisomerase inhibitor, an anti-metabolite, a mitotic inhibitor, an     alkylating agent, an anthracycline, a vinca alkaloid, an     intercalating agent, an agent capable of interfering with a signal     transduction pathway, an agent that promotes apoptosis, a proteosome     inhibitor or radiation. -   142) The method of embodiment 139, wherein the second therapeutic     agent is a vaccine. -   143) The method of embodiment 142, wherein the vaccine is a     polypeptide or fragment thereof, or a DNA or a RNA encoding the     polypeptide or fragment thereof expressed on tumor cells. -   144) The method of embodiment 143, wherein the polypeptide is PSMA,     mesothelin, EGFR or EGFRvIII. -   145) The method of embodiment 139, wherein the second therapeutic     agent is administered simultaneously, sequentially or separately. -   146) The method of any one of embodiments 135-145, wherein the     subject is treated or is being treated with radiation therapy. -   147) The method of any one of embodiments 135-145 wherein the     subject has had or will undergo surgery. -   148) The method of any one of embodiments 135-145, wherein the first     domain of the bispecific PSMA×CD3 antibody comprises the VH of SEQ     ID NO:66 and the VL or SEQ ID NO:67, and the second domain of the     bispecific PSMA×CD3 antibody comprises the VH of SEQ ID NO:104 and     the VL of SEQ ID NO:105. -   149) The antibody of any one of embodiments 109-126 for use in     therapy. -   150) An anti-idiotypic antibody binding to the antibody of any one     of embodiments 109-126.

Example 1: Materials

Generation of PSMA Cell Lines

Expression vectors presenting full-length chimpanzee PSMA (H2Q3K5_PANTR, SEQ ID NO: 1) or full length Cynomolgous monkey PSMA (EHH56646.1, SEQ ID NO: 2) were generated for use as screening tools to assess the anti-PSMA leads using an in-house expression vector with the CMV promoter using standard molecular biology techniques. Vectors were transiently transfected into HEK293F cells in suspension using standard methods. Transfected 293F suspension cells were plated in growth medium plus serum to become adherent and selected for stable plasmid integration. Single cell populations were selected by serial dilution and the PSMA surface receptor expression was quantified by FACS using the (PSMAL antibody (Center) affinity Purified Rabbit Polycolonal Antibody (Catalog # OAAB02483, Aviva Systems Biology) as the primary antibody with a R-PE anti-rabbit secondary antibody (Catalog #111-116-144, Jackson ImmunoResearch Laboratories, Inc.) and a rabbit polyclonal IgG (Catalog # SC-532, Santa Cruz Biotechnology) as the isotype control).

Human PSMA expressing cell lines were generated using lentivirus (Genecopoeia, cat # EX-G0050-Lv105-10) containing full length human PSMA (FOLH1_HUMAN, SEQ ID NO:3) and puromycin for selection of PSMA positive cells. HEK293F cells (ATCC), negative for PSMA, were transduced with Lentiviral particles to overexpress human PSMA. Following transduction, cells positively expressing PSMA and the resistance marker were selected by treating pooled cells, grown in DMEM+10% HI FBS (Life Technologies) and supplemented with varying concentrations of Puromycin (Life Technologies).

In addition to the HEK generated cell lines, several commercial cell lines were used for phage panning and binding and cellular toxicity assays. LNCaP clone FGC cells (ATCC cat # CRL-1740) are a commercially available human prostate cancer cell lines. C4-2B cells were originally developed at MD Anderson and are derived from LNCaP FGC grown in vivo and metastasize to bone marrow (Thalmann, et al 1994, Cancer Research 54, 2577-81).

Generation of Soluble PSMA ECD Proteins

Recombinant chimpanzee PSMA Extra Cellular Domain (ECD) protein (Chimp PSMA ECD, SEQ ID NO:4) was generated for panning and to assess the anti-PSMA leads using an in-house expression vector with the CMV promoter using standard molecular biology techniques. The chimp PSMA ECD gene fragment (amino acid 44-750 of SEQ ID NO: 1) with N-terminal signal sequence (SEQ ID NO:56), N-terminal Avitag (SEQ ID NO:57) and 6-His tags (SEQ ID NO:58) was cloned using an in-house expression vector with the CMV promoter using standard molecular biology techniques and transiently expressed in 293Expi cells (Invitrogen). cDNA was prepared using gene synthesis techniques (U.S. Pat. Nos. 6,670,127; 6,521,427). Supernatants were harvested and clarified by centrifugation. The proteins were purified using a two-step purification process: 1) IMAC purification with a HisTrap HP column (GE Healthcare) and 2) size exclusion purification (Superdex 200, Ge Healthcare) where the elution buffer is Dulbecco's phosphate-buffered saline, calcium, magnesium (Thermofisher, #14040) containing 0.5 mM ZnCl₂ to stabilize PSMA dimerization. Fractions containing the protein of interest were pooled and protein concentration was determined by A280. This material was used for binding and affinity measurements and is referred to as PSMG8.

Chimp PSMA ECD was also biotinylated for panning. The BirA plasmid that was co-transfected into mammalian cells to biotinylate proteins containing the Avi tag was created in-house. The BirA coding region (SEQ ID NO:59) was fused to the signal peptide from mouse IgG heavy chain (SEQ ID NO:80), and an ER retention signal (KDEL (“KDEL” disclosed as SEQ ID NO: 156) was added to the C-terminus to generate the BirA (SEQ ID NO:112). The constructed gene was cloned into an expression vector under the control of the CMV promoter. To produce biotinylated PSMA antigen, the PSMA plasmid DNA was added in a 4-fold excess (w/w) to the BirA plasmid into the transfection mix.

Biotinylation of the Chimp PSMA ECD protein was performed via the Avi tag by cotransfection of a BirA expression construct and the resulting secreted protein was purified using a two-step purification process: 1) IMAC purification with a HisTrap HP column (GE Healthcare) and 2) size exclusion purification (Superdex 200, Ge Healthcare) where the elution buffer is Dulbecco's phosphate-buffered saline, calcium, magnesium (Thermofisher, #14040) containing 0.5 mM ZnCl2 to stabilize PSMA dimerization. The protein was tested for endotoxin prior to use in phage panning studies.

Recombinant cynomolgous monkey PSMA extracellular domain (ECD) protein (cyno PSMA ECD, SEQ ID NO:5), corresponding to amino acids 44-750 of SEQ ID NO:2 with N-terminal signal (SEQ ID NO:56), N-terminal Avi-(SEQ ID NO:57) and 6His(SEQ ID NO:58) tags was cloned and expressed as described previously for the chimp PSMA ECD. Biotinylation of the cyno PSMA ECD protein was performed via the Avi tag by cotransfection of a BirA expression construct and the resulting secreted protein was purified by a two-step purification using IMAC HisTrap HP column (GE Healthcare) and MonoAvidin columns. The protein was tested for endotoxin prior to use in phage panning studies. This material was also used for binding and affinity measurements and is referred to as PSMG1.

A second recombinant cyno PSMA ECD protein (Cyno PSMA Fc, SEQ ID NO:6) with an IgG1 Fc (SEQ ID NO:81) was cloned and expressed using an in-house expression vector with the CMV promoter using standard molecular biology techniques. CynoPSMA Fc protein was transiently expressed in 293HEK-expi cells. Transient transfection of PSMG3 in HEK293 Expi cells were harvested 5 days after transfection, clarified by centrifugation (30 min, 6000 rpm) and filtered (0.2μ PES membrane, Corning). The relative amount of IgG was determined with the Octet instrument (ForteBio) using a purified known IgG (same isotype) spiked into spent medium to generate the standard curve.

Clarified Cyno PSMA Fc supernatant was loaded onto an equilibrated (dPBS, pH 7.2) HiTrap MabSelect Sure Protein A column (GE Healthcare) at a relative concentration of ˜30 mg protein per ml of resin. After loading, the column was washed with dPBS, pH7.2 and protein eluted with 10 column volumes of 0.1 M Na-Acetate, pH 3.5. Peak fractions were pooled, neutralized with 2M Tris, pH 7, and filtered (0.2p). The neutralized protein sample was dialyzed against 3 changes of dPBS containing Ca2+, Mg2+, and 0.5 mM ZnCl2, pH 7.2 overnight at 4° C. The next day, sample was removed from dialysis, filtered (0.2p) and the protein concentration determined by absorbance at 280 nm on a BioTek SynergyHT™ spectrophotometer. The quality of the purified proteins was assessed by SDS-PAGE and analytical size exclusion HPLC (Dionex HPLC system). Endotoxin levels were measured using a LAL assay (Pyrotell-T, Associates of Cape Cod). Purified proteins were stored at 4° C.

Recombinant human PSMA extracellular domain (ECD) protein (human PSMA ECD, SEQ ID NO:7), corresponding to amino acids 44-750 of SEQ ID NO:3 with N-terminal Avi- and 6His (SEQ ID NO: 58) tags was cloned, expressed and purified as described previously for the chimp and cyno PSMA ECD proteins.

Example 2: Identification of Anti-Chimp and Anti-Human PSMA Fabs

Panning with recombinant protein. A first solution panning of the de novo Human Fab-pIX libraries [Shi, L., et al J Mol Biol, 2010. 397(2): p. 385-396. WO 2009/085462], consisting of VH1-69, 3-23 and 5-51 heavy chain libraries paired with four human VL germline genes (A27, B3, L6, O12) libraries, was performed using an alternating panning approach with one round of phage capture on Strepavidin beads (Invitrogen Cat #112.05D, Lot #62992920) coated with biotinylated Chimp PSMA ECD according to the manufacturer's protocol, followed by phage capture on ProtG beads(Invitrogen, Cat #10003D) coated with Cyno-PSMA-Fc according to the manufacturer's protocol followed by phage capture on Sera-mag Double Speed magnetic Neutravidin beads (Thermo, Cat #7815-2104-011150) coated with biotinylated Chimp PSMA ECD according to the manufacturer's protocol. This panning yielded two hits: PSMB18 and PSMB25.

Whole cell panning for anti-PSMA Fabs. Additional panning experiments were performed on whole cells using the Round #1 output from the chimpanzee ECD panning experiments described above or fresh de novo phage libraries, as input. Briefly, phage was produced by helper phage infection and concentrated by PEG/NaCl precipitation according to standard protocols known in the art. The phage libraries were pre-cleared on untransfected parental HEK293F cells overnight at 4° C. with gentle rocking. Following PEG/NaCl precipitation, the pre-cleared libraries were incubated with chimp PSMA expressing HEK293 cells or LNCAP cells with gentle rocking for 2 hr at 4° C. The removal of unbound phage and the recovery of phage-bound cells was performed by Ficoll gradient, and following several wash steps with, cells carrying bound phage were incubated with 1 mL of TG-1 E. coli culture at 37° C. for 30 minutes without agitation. The resulting mixture was plated on LB-Carbenicillin-1% Glucose plates and grown over night at 37° C. The process was then repeated for subsequent panning rounds.

Conversion of phage Fab-pIX to Fab-His for generating E. coli supernatants. The resulting phage Fab-pIX hits were converted to Fab-His using a standard procedure. Plasmid DNA was isolated from phage panned E. coli (Plasmid Plus Maxi Kit, Qiagen cat #12963) and subjected to NheI/SpeI restriction digest. The resulting 5400 and 100 bp fragments were separated on a 0.8% agarose gel and the 5400 bp fragment was gel purified (MinElute PCR purification kit, Qiagen cat #28006). The purified 5400 bp band was self-ligated using T4 ligase and the resulting product (encoding the Fab-his fusion) was transformed back into the TG-1 E. coli strain and clonally isolated. Fab-His supernatants were generated from clones by overnight induction of cultures with 1 mM IPTG. Following centrifugation of the overnight culture, clarified supernatants were ready for use in downstream assays. To determine the relative expression levels of different Fab-his supernatants, an anti-kappa (Southern Biotech cat #2061-05) ELISA on serially diluted supernatants was performed. All of the clones tested exhibited similar Fab-his expression (data not shown).

Cell binding of Fab-his fusions from E. coli. A cell-based binding assay was designed to assess the binding capabilities of individual Fab-his fusions from E. coli supernatants to PSMA-expressing cells. Individual Fab clones were isolated from the round 3 output of all panning experiments following pIX excision. Fab clones were tested for binding to chimp and cyno PSMA expressing HEK cells, as well as to human PSMA on LNCaP cells. Briefly, PSMA expressing cells were aliquoted into a V-bottom plate (CoStar 3357) at a density of 200,000 per well and incubated with (100 μl) supernatants expressing Fab fragments for 1 hour on ice. Cells were washed twice with PBS containing 2% FBS, and stained with a mouse anti-human kappa-RPE antibody (Life Technologies cat # MH10514) for 1 hour on ice. Cells were washed twice with PBS containing 2% FBS and resuspended in 100 L of the same wash buffer. Plates were read on a BD FACS Array flow cytometer. FACS data was analyzed in FlowJo software by live gating the healthy population of cells using forward scatter and side scatter, and then analyzing the cells within this gate for PE staining. Mean fluorescence intensity (MFI) was calculated and exported into Microsoft Excel. Fab clones that exhibited binding≥3 times background for all three species of PSMA (cyno, chimp and human), and exhibited no binding to the HEK293 cell line, were labeled as “preliminary positive”. Fabs were sequenced and moved forward for cloning into mammalian expression vector for rescreening. True positives were selected from the binding of mammalian cell expressed Fab supernatants to PSMA-expressing cell lines.

Preparation of Mammalian Fabs. For conversion of E. coli Fab to mammalian-expressed Fab, In-Fusion HD cloning (ClonTech cat #638918) was utilized according to the manufacturer's protocol. Briefly, nucleotide sequences of clones that have passed the primary screen and are to be moved into mammalian Fab format, are loaded into the “InFu Primer Finder v1.2.3” program (software developed in-house), which generates a list of isotype-specific PCR primers used to generate PCR fragments for In-Fusion cloning into the huKappa_mulgGSP and huG1 Fab expression vectors. These vectors are in-house vectors with CMV promotors based off of pcDNA3.1. Following the In-fusion process, E. coli clones were isolated, sequence verified and transfected into HEK293 cells using standard protocols. Mammalian PSMA Fabs for confirming binding to PSMA expressing cell lines were prepared by harvesting 20 ml of supernatants from transfection after 5 days.

Rescreening hits from whole cell panning in mammalian sup format. Confirmation of mammalian expressed Fab supernatants was performed using the whole cell binding assay described previously. Binding of Fabs to Chimpanzee, Cynomolgous monkey and human PSMA (LNCaP cells) was tested, as well as counter screening for no binding to the parental HEK cell line. Table 3 shows the hit profile of mammalian Fab supernatant binding to PSMA-expressing cells. Many of the hits from E. coli supernatants did not confirm with mammalian expressed proteins. PSMB47 showed high binding to cyno PSMA-expressing cells and some binding to chimp-PSMA expressing cells, but no binding to LNCaP cells expressing human PSMA. PSMB55 showed a similar profile, but with some binding to LNCaP cells. PSMB68-PSMB79 bound to LNCaP cells, but not to chimp- or cyno-PSMA expressing cells. Mammalian Fab sups PSMB51, PSMB55 and PSMB56, bound all three cell lines. PSMB49, PSMB50, and PSMB53, show more chimp or cyno binding. M58 showed slight chimp and cyno binding.

TABLE 3 Hit profile of Mammalian Fab protein binding to PSMA-expressing cells measured by Geo-MFI (Mean Fluoresent Instensity) Fab protein ID (Fab DNA ID) cyno chimp LNCaP Parent HEK PSMB10 (PSMM10) 244 81.6 — 248 PSMB11 (PSMM11) 19 6.6 — 8.14 PSMB12 (PSMM12) 31.6 8.05 — 12.6 PSMB13 (PSMM13) 57.8 18.2 — 50.5 PSMB14 (PSMM14) 32.6 13.1 — 22.2 PSMB15 (PSMM15) 40.4 18.5 — 38 PSMB16 (PSMM16) 175 220 — 6.39 PSMB17 (PSMM17) 34.9 22.4 — 40.1 PSMB18 (PSMM18) 696 439 — 8.71 PSMB19 (PSMM19) 53.7 — 5.15 4.47 PSMB20 (PSMM20) 5.75 — 5.85 41.3 PSMB21 (PSMM21) 94.4 — 20.7 372 PSMB22 (PSMM22) 9.07 — 7.92 54.9 PSMB23 (PSMM23) 16.4 — 6.66 164 PSMB24 (PSMM24) 14.6 9.6 4.09 3.96 PSMB25 (PSMM25) 15.2 11.3 16.9 4.09 PSMB26 (PSMM26) 9.48 — 7.26 114 PSMB27 (PSMM27) 20 — 7.56 136 PSMB28 (PSMM28) 29.7 — 8.88 302 PSMB29 (PSMM29) 6.87 — 5.7 72.8 PSMB30 (PSMM30) 5.16 — 4.58 45 PSMB31 (PSMM31) 5.99 — — 25.5 PSMB32 (PSMM32) 4.81 — — 27.1 PSMB33 (PSMM33) 5.14 — — 40.1 PSMB34 (PSMM34) 17.9 — — 107 PSMB35 (PSMM35) 58.5 — — 231 PSMB36 (PSMM36) 5.05 — — 6.96 PSMB37 (PSMM37) 23.4 — — 178 PSMB38 (PSMM38) 4.05 — — 7.7 PSMB39 (PSMM39) 10.2 — — 166 PSMB40 (PSMM40) 66.9 — — 348 PSMB41 (PSMM41) 5.39 — — 12 PSMB42 (PSMM42) 7.35 — — 25.8 PSMB43 (PSMM43) 8.73 — — 7.18 PSMB44 (PSMM44) 12.6 — — 48.9 PSMB45 (PSMM45) 22.4 — — 43.1 PSMB46 (PSMM46) 3.88 — — 5.29 PSMB47 (PSMM48) 101 25.5 3.46 2.85 PSMB48 (PSMM49) 2.72 3.18 2.68 2.72 PSMB49 (PSMM50) 51.6 22 3.22 3.48 PSMB51 (PSMM52) 285 231 41.5 2.68 PSMB52 (PSMM53) 39.2 6.89 2.67 2.56 PSMB53 (PSMM54) 27.6 17.8 4 2.6 PSMB54 (PSMM55) 2.7 2.75 2.65 2.79 PSMB55 (PSMM56) 226 180 17.2 2.58 PSMB56 (PSMM57) 95.6 34.7 24.5 2.52 PSMB57 (PSMM58) 19.8 11 3.26 2.68 PSMB58 (PSMM59) 121 192 25.3 2.67 PSMB59 (PSMM60) 4.96 9.69 6.04 3 PSMB60 (PSMM61) 2.28 3.07 87.3 4.64 PSMB61 (PSMM62) 2.1 3.16 135 2.98 PSMB62 (PSMM63) 7.17 4.43 54.9 9.09 PSMB63 (PSMM64) 2.07 2.95 27 2.82 PSMB64 (PSMM65) 2.39 3.26 70.5 3.05 PSMB65 (PSMM66) 2.3 3.13 32.4 4.25 PSMB66 (PSMM67) 2.14 3 24.6 2.83 PSMB67 (PSMM68) 2.23 2.95 21 2.95 PSMB68 (PSMM69) 5.44 — 134 35.3 PSMB69 (PSMM70) 2.29 3.38 25.5 3.35 PSMB70 (PSMM71) 2.22 3.49 15.5 3.26 PSMB71 (PSMM72) 2.54 4.4 18.5 3.07 PSMB72 (PSMM73) 2.13 3.53 227 3.02 PSMB73 (PSMM74) 2.97 4.13 125 11.1 PSMB74 (PSMM75) 120 — 178 132 PSMB75 (PSMM76) 2.99 3.04 173 7.89 PSMB76 (PSMM77) 3.75 3.99 138 3.95 PSMB77 (PSMM78) 4.68 3.96 144 4.71 PSMB78 (PSMM79) 25.2 — 378 24.4 PSMB79 (PSMM80) 38.4 — 512 157 PSMB80 (PSMM81) 19.6 18.6 20.9 6.61 PSMB81 (PSMM82) 2.63 2.06 4.07 2.69 PSMB82 (PSMM83) 2.79 2.23 4.11 2.76 PSMB83 (PSMM84) 2.59 2.28 4.09 2.74 PSMB84 (PSMM85) 750 729 192 3.15 PSMB85 (PSMM86) 2.84 2.59 2.33 3.24 PSMB86 (PSMM87) 224 176 31.7 2.82 PSMB87 (PSMM88) 2.63 2.27 4.23 2.91 PSMB88 (PSMM89) 37.7 29.7 30.3 7.6 PSMB89 (PSMM90) 27.1 27.3 53.2 39.5 PSMB90 (PSMM91) 26.7 24.7 47.1 36.4 PSMB91 (PSMM92) 8.97 6.16 13 6.63 PSMB92 (PSMM93) 20 16.5 57.1 50 PSMB93 (PSMM94) 5.13 9.62 2.5 3.66 PSMB94 (PSMM95) 5.12 2.67 2.22 3.57 PSMB95 (PSMM96) 8.9 8.82 13.4 11.4 PSMB96 (PSMM97) 2.4 3.25 2.53 4.03 PSMB97 (PSMM98) 2.57 4.73 2.52 3.7 PSMB99 (PSMM100) 9.95 2.4 2.39 4.03 PSMB100 (PSMM101) 4.03 2.52 2.33 3.37 PSMB100 (PSMM101) 3.5 2.86 2.48 4.57 PSMB101 (PSMM102) 5.49 3.18 2.23 3.33 PSMB102 (PSMM103) 2.4 2.42 2.16 3.2 PSMB103 (PSMM104) 3.52 3.26 2.58 4.44 PSMB104 (PSMM105) 2.15 2.5 2.34 3.95 PSMB105 (PSMM106) 2.03 2.39 2.18 3.39 PSMB106 (PSMM107) 2 2.4 2.27 3.59 PSMB107 (PSMM108) 9 2.47 2.33 3.49 PSMB108 (PSMM109) 2 2.58 2.28 3.46 PSMB109 (PSMM110) 321 326 34.9 6.11 PSMB110 (PSMM111) 2.3 2.31 2.31 3.4 PSMB111 (PSMM112) 2.32 2.31 — 3.21 PSMB112 (PSMM113) 6.28 5.7 2.71 3.28 PSMB113 (PSMM114) 2.82 2.95 2.32 3.29 PSMB114 (PSMM115) 2.78 2.47 4.3 3.14 PSMB115 (PSMM116) 2.66 2.59 2.2 3.14 PSMB46 (PSMM117) 4.54 3.18 2.21 4.79 PSMB67 (PSMM118) 3.95 4.3 3 6.13 PSMB74 (PSMM119) 7.94 13 3.16 12.5 PSMB78 (PSMM120) 5.08 4.79 22.3 6.82 PSMB81 (PSMM121) 3.66 3.83 3.05 5.11 PSMB82 (PSMM122) 15.1 28.4 10.8 24.3 PSMB83 (PSMM123) 37.5 42.1 3.04 4.88 PSMB85 (PSMM124) 34.6 52.9 20.7 46.8 PSMB87 (PSMM125) 4.23 3.74 2.26 4.73 PSMB89 (PSMM126) 51.8 53.1 11.7 6.27 PSMB90 (PSMM127) 42.8 30.2 7.74 5.99 PSMB91 (PSMM128) 3.9 27.6 2.37 4.32 PSMB92 (PSMM129) 45.7 37.3 12.1 7.4 PSMB93 (PSMM130) 5.13 7.85 4.11 7.82 PSMB94 (PSMM131) 3.67 3.23 2.32 4.72 PSMB95 (PSMM132) 4.05 3.64 2.56 5.57 PSMB96 (PSMM133) 3.91 4.54 2.37 4.65 PSMB97 (PSMM134) 3.22 3.16 4.08 4.22 PSMB98 (PSMM135) 15.6 12.7 2.22 4.21 PSMB99 (PSMM136) 4.08 3.26 2.22 5.04 PSMB100 (PSMM137) 5.24 3.82 2.16 4.83 PSMB101 (PSMM138) 3.84 3.14 2.23 4.52 PSMB102 (PSMM139) 4.51 3.82 2.23 4.59 PSMB103 (PSMM140) 6.81 4.27 2.21 5.41 PSMB104 (PSMM141) 7.52 4.35 2.26 4.39 PSMB105 (PSMM142) 5.03 11.2 4.87 7.28 PSMB106 (PSMM143) 3.87 3.8 2.73 4.9 PSMB107 (PSMM144) 3.3 3.35 2.3 4.64 PSMB108 (PSMM145) 6.78 3.83 2.33 4.98 PSMB110 (PSMM146) 4.03 3.23 2.28 5.3 PSMB111 (PSMM147) 3.71 3.26 2.36 5.11 PSMB112 (PSMM148) 4.54 3.26 2.26 4.86 PSMB113 (PSMM149) 84.3 104 51.7 94.2 PSMB114 (PSMM150) 3.31 3.26 2.21 5.14 PSMB115 (PSMM151) 3.55 3.43 2.3 4.21

Dose response curves of mammalian expressed Fabs. Once mammalian expressed Fab clones were confirmed for positive binding as neat Fab supernatants to PSMA expressing cell lines, the supernatants were normalized for protein concentration by Octet or protein gel, and dose-response curves were completed to confirm PSMA binding using the protocol described previously. FIGS. 1-3 show titration curves for hits that demonstrated binding to all three PSMA-expressing cells. FIGS. 1A-D show the titration curves for anti-PSMA panning hits vs. LNCaP cells. FIGS. 2A-D show the titration curves for anti-PSMA panning hits vs Chimp-PSMA HEK cells. FIGS. 3A-D show the titration curves for anti-PSMA panning hits vs Cyno-PSMA HEK cells. PSMG5 (Cyno PSMA, GenBank: EHH56646.1) or PSMG9 (Chimp PSMA, NCBI Reference Sequence: XP_016777253.1) were cloned into a mammalian expression vector between HindII and EcoRI sites under control of CMV promoter for cell line generation. Engineered DNA was transfected into 293F cells using lipofectamine LTX reagent followed by geneticin selection to select for PSMA (PSMG5 or PSMG9) positive cells. Following selection, cells were screened and sorted using anti-PSMA antibody (Aviva Cat # OAAB02483-PSMG9) or anti-PSMA Fab (PSMB18-Janssen Internal) using FACS. FACS sorted PSMG5 clones 11, 23, 25 and 32 and PSMG9 clones 2, 10, 11, 12, 20 and 24 were selected and handed off for screening. PSMG5 and PSMG9 sequences are provided below. Binding profiles among hits were compared across cell lines expressing different species of PSMA. PSMB51 supernatant was used as a positive control across experiments. Several hits were deprioritized because of N-linked glycosylation sites in CDRs, binding to the PSMA negative parental HEK cell line, or lack of binding to PSMA positive cell lines. Eleven Fab hits remained and 10 hits were cloned into human IgG4-PAA heavy chain constructs and used to generate PSMA×CD3 bispecific antibodies. These hits showed cross-species binding within 3-fold of each other and were moved into a bispecific antibody format to be tested for T cell redirection killing of PSMA positive targets. The panning antigens for each hit is shown in Table 4.

PSMG5 (SEQ ID NO: 126) MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSSE ATNITPKHNMKAFLDELKAENIKKFLHNFTQIPHLAGTEQNFQLAKQIQS QWKEFGLDSVELTHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPP AGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSG KIVIARYGKVFRGNKVKNAQLAGATGVILYSDPDDYFAPGVKSYPDGW NLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGMAEAVGLPSIPV HPIGYYDAQKLLEKMGGSASPDSSWRGSLKVPYNVGPGFTGNFSTQKV KMHIHSTSEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSG AAVVHEIVRSFGMLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENS RLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVYNLTKELESPDEGF EGKSLYESTWKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYT KNWETNKFSSYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVF ELANSVVLPFDCRDYAVVLRKYADKIYNISMKHPQEMKTYSVSFDSLFS AVKNFTEIASKFSERLRDFDKSNPILLRMMNDQLMFLERAFIDPLGLPDR PFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSQAWGEVKRQISI ATFTVQAAAETLSEVA PSMG9 (SEQ ID NO: 127) MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEA TNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQ WKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPP GYENVLDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGK IVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLP GGGVQRGNILNLNGAGDPLTPGYPANEYAYRHGIAEAVGLPSIPVHPIGY YDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHST NEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIV RSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAY INADSSIEGNYTLRVDCTPLMYSLVYNLTKELKSPDEGFEGKSLYESWTK KSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGY PLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRD YAVVLRKYADKIYNISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFTER LQDFDKSNPILLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNK YAGESFPGIYDALFDIESKVDPSKAWGDVKRQISVAAFTVQAAAETLSEV A

TABLE 4 Antigen for each of the panning hits Round 1 antigen Round 2-3 antigen Hits Hit identification Chimp PSMA ECD Cyno PSMA ECD 2 PSMB18, PSMB25 Chimp PSMA ECD Chimp PSMA HEK 9 PSMB49, PSMB51, PSMB56, PSMB58, PSMB109, PSMB55, PSMB84, PSMB83 LNCaP Chimp PSMA HEK 2 PSMB86, PSMB80

Preparation of anti-PSMA mAbs. A total of 12 clones that demonstrated binding to all three PSMA-expressing cells were ultimately converted to mAb IgG4 having Fc substitutions S228P, F234A, and L235A (PAA) isotype by restriction cloning. Briefly, constructs corresponding to Fab clones that have passed initial screens were digested with HindIII and ApaI. Gel purified fragments were ligated into an in-house expression vector with CMV promoter for generation of human IgG4-PAA expression. This allowed for rapid generation of bispecific antibodies. The in-house expression vector previously described was used to express the Heavy and Light Chains for each PSMA mab, where both vectors were co-transfected transiently into 293Expi or CHO cell lines for expression of the mAb. CDR sequences of cross-species positive PSMA Fabs generated from phage panning are shown below in Table 5. VH and VL sequences of the selected Fabs are shown below in Table 6. Heavy and light chain sequences of mAbs generated from the Fabs are shown in Table 7.

TABLE 5 CDR sequences (defined according to Kabat)  of FAbs from phage panning (corresponding  SEQ ID NOs are listed in parentheses) CDRs (SEQ ID NO:) FAB ID CDR1 CDR2 CDR3 PSMB58 HC SYWIS IIPGDSYTR DYEWELFDSR (122) YSPSFQG LDY (123) (124) LC RASQSISSYLN AASSLQS QQSYSTP (23) (12) (24) PSMB109 HC NAWIS WINPESGRA ELYYLVYSTY (8) NYAQKFQG YYAFDY (9) (10) LC RASQSIDRWLN AASSLQS QQSPRYPLT (11) (12) (13) PSMB86 HC SYDIS GIIPIEGTA DYPAGYGFDY (14) NYAQKFQG (16) (15) LC RASQSVSSSYL GASSRAT QQYGSSPLT A (18) (19) (17) PSMB84 HC SDWMS AISGNGGST DPYYYYDGDS (20) EYADSVKG YYGMDV (21) (22) LC RASQSISSYLN AASSLQS QQSYSTP (23) (12) (24) PSMB83 HC SDAMH EISGSGGYT DSYDSSLYVG (25) NYADSVKG DYFDY (26) (27) LC RASQSVSSYLA DASNRAT QQRSNWPLT (28) (29) (30) PSMB56 HC SYAIS WISPYNGNA DSDRSYNLDY (31) NYAQKFQG (33) (32) LC RASQSISGWLN AASSLQS QQSYSTPLT (34) (12) (35) PSMB55 HC SYWIG IIYPGDSDT GLPIWYLDY (36) RYSPSFQG (38) (37) LC RASQSVASDLA FASNRAT QQSITWPFT (39) (40) (41) PSMB51 HC SYAIS WIIPYNGNA VNSAALVWER (31) NYAQKFQG LDY (42) (43) LC RASQSIDRWLN AASSLQS QQSPRYPLT (11) (12) (13) PSMB49 HC SYAIS GIIPIFGTA ASRVMHASYG (31) NYAQKFQG YLDY (44) (45) LC RASQSVSKWLA DASNRAT QQRFTAPWT (46) (29) (47) PSMB25 HC SYWIG IIYPGDSDT GWAYDRGLDY (36) RYSTSFQG (48) (37) LC KSSQSVLYSSN WASTRES QQYYSTPLT NKNYLA (50) (51) (49) PSMB18 HC SYWIG IIYPGDSDT AYHYSKGLDY (36) RYSPSFQG (52) (37) LC KSSQSVLYSSN WASTRES QQYYSTPLT NKNYLA (50) (51) (49) PSMB80 HC DYAIS RIDPIEGTA DRYYYDGVYW (53) NYAQKFQG YSDYFDY (54) (55) LC RASQSISSYLN AASSLQS QQSYSTPLT (23) (12) (35)

A monospecific anti-PSMA antibody PSMB119 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 79 and the VL of SEQ ID NO: 78 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB120 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 77 and the VL of SEQ ID NO: 78 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB121 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 75 and the VL of SEQ ID NO: 76 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB122 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 74 and the VL of SEQ ID NO: 61 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB123 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 72 and the VL of SEQ ID NO: 73 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB124 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 70 and the VL of SEQ ID NO: 71 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB126 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 68 and the VL of SEQ ID NO: 69 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB127 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 66 and the VL of SEQ ID NO: 67 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB128 (Alt. Fab ID: PSMB84) was generated comprising the VH and VL regions having the VH of SEQ ID NO: 64 and the VL of SEQ ID NO: 65 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB129 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 60 and the VL of SEQ ID NO: 61 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB130 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 62 and the VL of SEQ ID NO: 63 and an IgG4 constant region with S228P, F234A, and L235A substitutions.

TABLE 6 VH and VL sequences of PSMA Fabs SEQ SEQ FAB VH Ammo acid ID VL Amino Acid ID ID sequence NO Sequence NO PSMB QVQLVQSGAEVKKPGSSV  60 DIQMTQSPSSLSASVGDRV 61 109 KVSCKASGGTFSSYAISW TITCRASQSIDRWLNWYQQ VRQAPGQGLEWMGWISPY KPGKAPKLLIYAASSLQSG NGNANYAQKFQGRVTITA VPSRESGSGSGTDFTLTIS DESTSTAYMELSSLRSED SLQPEDFATYYCQQSPRYP TAVYYCARVNSAALVWER LTFGQGTKVEIK LDYWGQGTLVTVSS PSMB86 QVQLVQSGAEVKKPGSSV  62 EIVLTQSPGTLSLSPGERA 63 KVSCKASGGTFKSYDISW TLSCPASQSVSSSYLAWYQ VRQAPGQGLEWMGGIIPI QKPGQAPRLLIYGASSRAT EGTANYAQKFQGRVTITA GIPDRFSGSGSGTDFTLTI DESTSTAYMELSSLRSED SRLEPEDFAVYYCQQYGSS TAVYYCARDYPAGYGFDY PLTFGQGTKVEIK WGQGTLVTVSS PSMB84 EVQLLESGGGLVQPGGSL  64 DIQMTQSPSSLSASVGDRV 65 RLSCAASGFTFDSDWMSW TITCRASQSISSYLNWYQQ VRQAPGKGLEWVSAISGN KPGKAPKLLIYAASSLQSG GGSTEYADSVKGRFTISR VPSRFSGSGSGTDFTLTIS DNSKNTLYLQMNSLRAED SLQPEDFATYYCQQSYSTP TAVYYCARDPYYYYDGDS LTFGQGTKVEIK YYGMDVWGQGTLVTVSS PSMB83 EVQLLESGGGLVQPGGSL  66 EIVITQSPATLSLSPGERA 67 RLSCAASGFTFKSDAMHW TLSCRASQSVSSYLAWYQQ VRQAPGKGLEWVSEISGS KPGQAPPLLIYDASNRATG GGYTNYADSVKGRFTISR IPARFSGSGSGTDFTLTIS DNSKNTLYLQMNSLRAED SLEPEDFAVYYCQQRSNWP TAVYYCARDSYDSSLYVG LTFGQGTKVEIK DYEDYWGQGTLVTVSS PSMB80 QVQLVQSGAEVKKPGSSV  68 DIQMTQSPSSLSASVGDRV 69 KVSCKASGGTFDDYAISW TITCRASQSISSYLNWYQQ VRQAPGQGLEWMGRIDPI KPGKAPKLLIYAASSLQSG EGTANYAQKFQGRVTITA VPSRFSGSGSGTDFTLTIS DESTSTAYMELSSLRSED SLQPEDFATYYCQQSYSTP TAVYYCARDRYYYDGVYW LTFGQGTKVEIK YSDYFDYWGQGTLVTVSS PSMB58 EVQLVQSGAEVKKPGESL 160 DIQMTQSPSSLSASVGDRV 65 KISCKGSGYSFTSYWISW TITCRASQSISSYLNWYQQ VRQMPGKGLEWMGIIYPG KPGKAPKLLIYAASSLQSG DSYTRYSPSFQGQVTISA VPSRFSGSGSGTDFTLTIS DKSISTAYLQWSSLKASD SLQPEDFATYYCQQSYSTP TAMYYCARDYEWELFDSR LTFGQGTKVEIK LDYWGQGTLVTVSS PSMB56 QVQLVQSGAEVKKPGSSV  70 DIQMTQSPSSLSASVGDRV 71 KVSCKASGGTFSSYAISW TITCRASQSISGWLNWYQQ VRQAPGQGLEWMGWISPY KPGKAPKLLIYAASSLQSG NGNANYAQKFQGRVTITA VPSRFSGSGSGTDFTLTIS DESTSTAYMELSSLRSED SLQPEDFATYYCQQSYSTP TAVYYCARDSDRSYNLDY LTFGQGTKVEIK WGQGTLVTVSS PSMB55 EVQLVQSGAEVKKPGESL  72 EIVLTQSPATLSLSPGERA 73 KISCKGSGYSFTSYWIGW TLSCRASQSVASDLAWYQQ VRQMPGKGLEWMGIIYPG KPGQAPRLLIYFASNRATG DSDTRYSPSFQGQVTISA IPARFSGSGSGTDFTLTIS DKSISTAYLQWSSLKASD SLEPEDFAVYYCQQSITWP TAMYYCARGLPIWYLDYW FTFGQGTKVEIK GQGTLVTVSS PSMB51 QVQLVQSGAEVKKPGSSV  74 DIQMTQSPSSLSASVGDRV 61 KVSCKASGGTFSSYAISW TITCRASQSIDRWLNWYQQ VRQAPGQGLEWMGWIIPY KPGKAPKLLIYAASSLQSG NGNANYAQKFQGRVTITA VPSRFSGSGSGTDFTLTIS DESTSTAYMELSSLRSED SLQPEDFATYYCQQSPRYP TAVYYCARVNSAALVWER LTFGQGTKVEIK LDYWGQGTLVTVSS PSMB49 QVQLVQSGAEVKKPGSSV  75 EIVLTQSPATLSLSPGERA 76 KVSCKASGGTFSSYAISW TLSCRASQSVSKWLAWYQQ VRQAPGQGLEWMGGIIPI KPGQAPRLLIYDASNRATG FGTANYAQKFQGRVTITA IPARFSGSGSGTDFTLTIS DESTSTAYMELSSLRSED SLEPEDFAVYYCQQRFTAP TAVYYCARASRVWHASYG WTFGQGTKVEIK YLDYWGQGTLVTVSS PSMB25 EVQLVQSGAEVKKPGESL  77 DIVMTQSPDSLAVSLGERA 78 KISCKGSGYSFTSYWIGW TINCKSSQSVLYSSNNKNY VRQMPGKGLEWMGIIYPG LAWYQQKPGQPPKLLIYWA DSDTRYSPSFQGQVTISA STRESGVPDRFSGSGSGTD DKSISTAYLQWSSLKASD FTLTISSLQAEDVAVYYCQ TAMYYCARGWAYDRGLDY QYYSTPLTFGQGTKVEIK WGQGTLVTVSS PSMB18 EVQLVQSGAEVKKPGESL  79 DIVMTQSPDSLAVSLGERA 78 KISCKGSGYSFTSYWIGW TINCKSSQSVLYSSNNKNY VRQMPGKGLEWMGIIYPG LAWYQQKPGQPPKLLIYWA DSDTRYSPSFQGQVTISA STRESGVPDRFSGSGSGTD DKSISTAYLQWSSLKASD FTLTISSLQAEDVAVYYCQ TAMYYCARAYHYSKGLDY QYYSTPLTFGQGTKVEIK WGQGTLVTVSS

TABLE 7 Heavy and Light chain sequences of Mabs with corresponding SEQ ID NOs SEQ SEQ Heavy Chain Amino ID Light Chain Amino ID mAb ID acid sequence NO Acid Sequence NO PSMB129 QVQLVQSGAEVKKPGSSVKVSCKA  82 DIQMTQSPSSLSASVGDRVTIT  83 (FAB SGGTFSSYAISWVRQAPGQGLEWM CRASQSIDRWLNWYQQKPGKAP PSMB109) GWISPYNGNANYAQKFQGRVTITA KLLIYAASSLQSGVPSRFSGSG DESTSTAYMELSSLRSEDTAVYYC SGTDFTLTISSLQPEDFATYYC ARVNSAALVWERLDYWGQGTLVTV QQSPRYPLTFGQGTKVEIKRTV SSASTKGPSVFPLAPCSRSTSEST AAPSVFIFPPSDEQLKSGTASV AALGCLVKDYFPEPVTVSWNSGAL VCLLNNFYPREAKVQWKVDNAL TSGVHTFPAVLQSSGLYSLSSVVT QSGNSQESVTEQDSKDSTYSLS VPSSSLGTKTYTCNVDHKPSNTKV STLTLSKADYEKHKVYACEVTH DKRVESKYGPPCPPCPAPEAAGGP QGLSSPVTKSFNRGEC SVFLFPPKPKDTLMISRTPEVTCV VVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK PSMB130 QVQLVQSGAEVKKPGSSVKVSCKA  84 EIVLTQSPGTLSLSPGERATLS  85 (FAB SGGTFKSYDISWVRQAPGQGLEWM CRASQSVSSSYLAWYQQKPGQA PSMB86) GGIIPIEGTANYAQKFQGRVTITA PRLLIYGASSRATGIPDRFSGS DESTSTAYMELSSLRSEDTAVYYC GSGTDFTLTISRLEPEDFAVYY ARDYPAGYGFDYWGQGTLVTVSSA CQQYGSSPLTFGQGTKVEIKRT STKGPSVFPLAPCSRSTSESTAAL VAAPSVFIFPPSDEQLKSGTAS GCLVKDYFPEPVTVSWNSGALTSG VVCLLNNFYPREAKVQWKVDNA VHTFPAVLQSSGLYSLSSVVTVPS LQSGNSQESVTEQDSKDSTYSL SSLGTKTYTCNVDHKPSNTKVDKR SSTLTLSKADYEKHKVYACEVT VESKYGPPCPPCPAPEAAGGPSVF HQGLSSPVTKSFNRGEC LFPPKPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKN QVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PSMB128 EVQLLESGGGLVQPGGSLRLSCAA  86 DIQMTQSPSSLSASVGDRVTIT  87 (FAB SGFTFDSDWMSWVRQAPGKGLEWV CRASQSISSYLNWYQQKPGKAP PSMB84) SAISGNGGSTEYADSVKGRFTISR KLLIYAASSLQSGVPSRFSGSG DNSKNTLYLQMNSLRAEDTAVYYC SGTDFTLTISSLQPEDFATYYC ARDPYYYYDGDSYYGMDVWGQGTL QQSYSTPLTFGQGTKVEIKRTV VTVSSASTKGPSVFPLAPCSRSTS AAPSVFIFPPSDEQLKSGTASV ESTAALGCLVKDYFPEPVTVSWNS VCLLNNFYPREAKVQWKVDNAL GALTSGVHTFPAVLQSSGLYSLSS QSGNSQESVTEQDSKDSTYSLS VVTVPSSSLGTKTYTCNVDHKPSN STLTLSKADYEKHKVYACEVTH TKVDKRVESKYGPPCPPCPAPEAA QGLSSPVTKSFNRGEC GGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK PSMB127 EVQLLESGGGLVQPGGSLRLSCAA  88 EIVLTQSPATLSLSPGERATLS  89 (FAB SCFTFKSDAMHWVRQAPGKGLEWV CRASQSVSSYLAWYQQKPGQAP PSMB83) SEISGSGGYTNYADSVKGRFTISR RLLIYDASNRATGIPARFSGSG DNSKNTLYLQMNSLRAEDTAVYYC SGTDFTLTISSLEPEDFAVYYC ARDSYDSSLYVGDYFDYWGQGTLV QQRSNWPLTFGQGTKVEIKRTV TVSSASTKGPSVFPLAPCSRSTSE AAPSVFIFPPSDEQLKSGTASV STAALGCLVKDYFPEPVTVSWNSG VCLLNNFYPREAKVQWKVDNAL ALTSGVHTFPAVLQSSGLYSLSSV QSGNSQESVTEQDSKDSTYSLS VTVPSSSLGTKTYTCNVDHKPSNT STLTLSKADYEKHKVYACEVTH KVDKRVESKYGPPCPPCPAPEAAG QGLSSPVTKSFNRGEC GPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQE EMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPVVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK PSMB127 atggcttgggtgtggaccttgc 161 atggcctgggtgtggaccct 162 DNA tattcctgatggcagctgccca gctgttcctgatggccgccg aagtatacaggccgaggttcag cccagagcatccaggccgag ctgctggaatctggcggaggat atcgtgctgacccagagccc tggttcagcctggcggctctct cgccaccctgagcctgagcc gagactgtcttgtgccgcttct ccggcgagcgggccaccctg ggcttcaccttcaagtccgacg agctgccgggccagccagag ctatgcactgggtccgacaggc cgtgagcagctacctggcct ccctggaaaaggactggaatgg ggtaccagcagaagcccggc gtgtccgagatctctggctctg caggccccccggctgctgat gcggctacaccaactacgccga ctacgacgccagcaaccggg ctccatgaagtcccggttcacc ccaccggcatccccgcccgg atctctcgggacaactccaaga ttcagcggcagcggcagcgg acaccctgtacctgcagatgaa caccgacttcaccctgacca ctccctgagagccgaggacacc tcagcagcctggagcccgag gccgtgtactactgcgccagag gacttcgccgtgtactactg actcctacgactccagcctgta ccagcagcggagcaactggc cgtgggcgactacttagattat ccctgaccttcggccagggc tggggccagggcaccctggtca accaaggtggagatcaagcg ccgtttcttctgcttccaccaa tacggtggctgcaccatctg gggcccatccgtcttccccctg tcttcatcttcccgccatct gcgccctgctccaggagcacct gatgagcagttgaaatctgg ccgagagcacagccgccctggg aactgcctctgttgtgtgcc ctgcctggtcaaggactacttc tgctgaataacttctatccc cccgaaccggtgacggtgtcgt agagaggccaaagtacagtg ggaactcaggcgccctgaccag gaaggtggataacgccctcc cggcgtgcacaccttcccggct aatcgggtaactcccaggag gtcctacagtcctcaggactct agtgtcacagagcaggacag actccctcagcagcgtggtgac caaggacagcacctacagcc cgtgccctccagcagcttgggc tcagcagcaccctgacgctg acgaaaacctacacctgcaacg agcaaagcagactacgagaa tagatcacaagcccagcaacac acacaaagtctacgcctgcg caaggtggacaagagagttgag aagtcacccatcagggcctg tccaaatatggtcccccatgcc agctcgcccgtcacaaagag caccatgcccagcacctgaggc cttcaacaggggagagtgt cgccgggggaccatcagtcttc ctgttccccccaaaacccaagg acactctcatgatctcccggac ccctgaggtcacgtgcgtggtg gtggacgtgagccaggaagacc ccgaggtccagttcaactggta cgtggatggcgtggaggtgcat aatgccaagacaaagccgcggg aggagcagttcaacagcacgta ccgtgtggtcagcgtcctcacc gtcctgcaccaggactggctga acggcaaggagtacaagtgcaa ggtctccaacaaaggcctcccg tcctccatcgagaaaaccatct ccaaagccaaagggcagccccg agagccacaggtgtacaccctg cccccatcccaggaggagatga ccaagaaccaggtcagcctgac ctgcctggtcaaaggcttctac cccagcgacatcgccgtggagt gggagagcaatgggcagccgga gaacaactacaagaccacgcct cccgtgctggactccgacggct ccttcttcctctacagcaggct aaccgtggacaagagcaggtgg caggaggggaatgtcttctcat gctccgtgatgcatgaggctct gcacaaccactacacacagaag agcctctccctgtctctgggta aa PSMB126 QVQLVQSGAEVKKPGSSVKVSCKA  90 DIQMTQSPSSLSASVGDRVTIT  91 (FAB SGGTFDDYAISWVRQAPGQGLEWM CRASQSISSYLNWYQQKPGKAP PSMB80) GRIDPIEGTANYAQKFQGRVITA KLLIYAASSLQSGVPSRESGSG DESTSTAYMELSSLRSEDTAVYYC SGTDFTLTISSLQPEDFATYYC ARDRYYYDGVYWYSDYFDYWGQGT QQSYSTPLTFGQGTKVEIKRTV LVTVSSASTKGPSVFPLAPCSRST AAPSVFIFPPSDEQLKSGTASV SESTAALGCLVKDYFPEPVTVSWN VCLLNNFYPREAKVQWKVDNAL SGALTSGVHTFPAVLQSSGLYSLS QSGNSQESVTEQDSKDSTYSLS SVVTVPSSSLGTKTYTCNVDHKPS STLTLSKADYEKHKVYACEVTH NTKVDKRVESKYGPPCPPCPAPEA QGLSSPVTKSFNRGEC AGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGV EVHNAKTKPREEQFNSTYRVVSVL TVLHQDWLNGKEYKCKVSNRGLPS SIERTISKAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIA VEWESNEQPENNYKTTPPVLDSDG SFELYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGK PSMB124 QVQLVQSGAEVKKPGSSVKVSCKA  92 DIQMTQSPSSLSASVGDRVTIT  93 (FAB SGGTFSSYAISWVRQAPGQGLEWM CRASQSISGWLNWYQQKPGKAP PSMB56) GWISPYNGNANYAQKFQGRVTITA KLLIYAASSLQSGVPSRFSGSG DESTSTAYMELSSLRSEDTAVYYC SGTDFTLTISSLQPEDFATYYC ARDSDRSYNLDYWGQGTLVTVSSA QQSYSTPLTFGQGTKVEIKRTV STKGPSVFPLAPCSRSTSESTAAL AAPSVFIFPPSDEQLKSGTASV GCLVKDYFPEPVTVSWNSGALTSG VCLLNNFYPREAKVQWKVDNAL VHTFPAVLQSSGLYSLSSVVTVPS QSGNSQESVTEQDSKDSTYSLS SSLGTKTYTCNVDHKPSNTKVDKR STLTLSKADYEKHKVYACEVTH VESKYGPPCPPCPAPEAAGGPSVF QGLSSPVTKSFNRGEC LFPPKPKDTLMISPTPEVTCVVVD VSQEDPEVQFNWYVDEVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKN QVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALH NHYTQRSLSLSLGK PSMB123 EVQLVQSGAEVKKPGESLKISCKG  94 EIVLTQSPATLSLSPGERATLS  95 (FAB SGYSFTSYWIGWVRQMPGKGLEWM CRASQSVASDLAWYQQKPGQAP PSMB55) GIIYPGDSDTRYSPSFQGQVTISA RLLIYFASNRATGIPARFSGSG DKSISTAYLQWSSLKASDTAMYYC SGTDFTLTISSLEPEDFAVYYC ARGLPIWYLDYWGQGTLVTVSSAS QQSITWPFTFGQGTKVEIKRTV TKGPSVFPLAPCSRSTSESTAALG AAPSVFIFPPSDEQLKSGTASV CLVKDYFPEPVTVSWNSGALTSGV VCLLNNFYPREAKVQWKVDNAL HTFPAVLQSSGLYSLSSVVTVPSS QSGNSQESVTEQDSKDSTYSLS SLGTKTYTCNVDHKPSNTKVDKRV STLTLSKADYEKHKVYACEVTH ESKYGPPCPPCPAPEAAGGPSVFL QGLSSPVTKSFNRGEC FPPKPKDTLMISRTPEVTCVVVDV SQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGK PSMB122 QVQLVQSGAEVKKPGSSVKVSCKA  96 DIQMTQSPSSLSASVGDRVTIT  83 (FAB SGGTFSSYAISWVRQAPGQGLEWM CRASQSIDRWLNWYQQKPGKAP PSMB51) GWIIPYNGNANYAQKFQGRVTITA KLLIYAASSLQSGVPSRFSGSG DESTSTAYMELSSLRSEDTAVYYC SGTDFTLTISSLQPEDFATYYC ARVNSAALVWERLDYWGQGTLVTV QQSPRYPLTFGQGTKVEIKRTV SSASTKGPSVFPLAPCSRSTSEST AAPSVFIFPPSDEQLKSGTASV AALGCLVKDYFPEPVTVSWNSGAL VCLLNNFYPREAKVQWKVDNAL TSGVHTFPAVLQSSGLYSLSSVVT QSGNSQESVTEQDSKDSTYSLS VPSSSLGTKTYTCNVDHKPSNTKV STLTLSKADYEKHKVYACEVTH DKRVESKYGPPCPPCPAPEAAGGP QGLSSPVTKSFNRGEC SVFLFPPKPKDTLMISRTPEVTCV VVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK PSMB121 QVQLVQSGAEVKKPGSSVKVSCKA  97 EIVLTQSPATLSLSPGERATLS  98 (FAB SGGTFSSYAISWVRQAPGQGLEWM CRASQSVSKWLAWYQQKPGQAP PSMB49) GGIIPIFGTANYAQKFQGPVTITA RLLIYDASNRATGIPARFSGSG DESTSTAYMELSSLRSEDTAVYYC SGTDFTLTISSLEPEDFAVYYC ARASRVWHASYGYLDYWGQGTLVT QQRFTAPWTFGQGTKVEIKRTV VSSASTKGPSVFPLAPCSRSTSES AAPSVFIFPPSDEQLKSGTASV TAALGCLVKDYFPEPVTVSWNSGA VCLLNNFYPREAKVQWKVDNAL LTSGVHTFPAVLQSSGLYSLSSVV QSGNSQESVTEQDSKDSTYSLS TVPSSSLGTKTYTCNVDHKPSNTK STLTLSKADYEKHKVYACEVTH VDKRVESKYGPPCPPCPAPEAAGG QGLSSPVTKSFNRGEC PSVFLFPPKPKDTLMISRTPEVTC VVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGOPREPQVYTLPPSQEE MTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGK PSMB120 EVQLVQSGAEVKKPGESLKISCKG  99 DIVMTQSPDSLAVSLGERATIN 100 (FAB SGYSFTSYWIGWVRQMPGKGLEWM CKSSQSVLYSSNNKNYLAWYQQ PSMB25) GIIYPGDSDTRYSPSFQGQVTISA KPGQPPKLLIYWASTRESGVPD DKSISTAYLQWSSLKASDTAMYYC RFSGSGSGTDFTLTISSLQAED ARGWAYDRGLDYWGQGTLVTVSSA VAVYYCQQYYSTPLTFGQGTKV STKGPSVFPLAPCSRSTSESTAAL EIKRTVAAPSVFIFPPSDEQLK GCLVKDYFPEPVTVSWNSGALTSG SGTASVVCLLNNFYPREAKVQW VHTFPAVLQSSGLYSLSSVVTVPS KVDNALQSGNSQESVTEQDSKD SSLGTKTYTCNVDHKPSNTKVDKR STYSLSSTLTLSKADYEKHKVY VESKYGPPCPPCPAPEAAGGPSVF ACEVTHQGLSSPVTKSFNRGEC LFPPKPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVEVHNAKT NPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKN QVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PSMB119 EVQLVQSGAEVKKPGESLKISCKG 101 DIVMTQSPDSLAVSLGERATIN 100 (FAB SGYSFTSYWIGWVRQMPGKGLEWM CKSSQSVLYSSNNKNYLAWYQQ PSMB18) GIIYPGDSDTRYSPSFQGQVTISA KPGQPPKLLIYWASTRESGVPD DKSISTAYLQWSSLKASDTAMYYC RFSGSGSGTDFTLTISSLQAED ARAYHYSKGLDYWGQGTLVTVSSA VAVYYCQQYYSTPLTFGQGTKV STKGPSVFPLAPCSRSTSESTAAL EIKRTVAAPSVFIFPPSDEQLK GCLVKDYFPEPVTVSWNSGALTSG SGTASVVCLLNNFYPREAKVQW VHTFPAVLQSSGLYSLSSVVTVPS KVDNALQSGNSQESVTEQDSKD SSLGTKTYTCNVDHKPSNTKVDKR STYSLSSTLTLSKADYEKHKVY VESKYGPPCPPCPAPEAAGGPSVF ACEVTHQGLSSPVTKSFNRGEC LFPPKPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKN QVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PSMB87 EVQLVQSGAEVKKPGESLKISCKG 125 DIQMTQSPSSLSASVGDRVTIT  91 (FAB SGYSFTSYWISWVRQMPGKGLEWM CRASQSISSYLNWYQQKPGKAP PSMB58) GIIYPGDSYTRYSPSFQGQVTISA KLLIYAASSLQSGVPSRFSGSG DKSISTAYLQWSSLKASDTAMYYC SGTDFTLTISSLQPEDFATYYC ARDYEWELFDSRLDYWGQGTLVTV QQSYSTPLTFGQGTKVEIKRTV SSASTKGPSVFPLAPCSRSTSEST AAPSVFIFPPSDEQLKSGTASV AALGCLVKDYFPEPVTVSWNSGAL VCLLNNFYPREAKVQWKVDNAL TSGVHTFPAVLQSSGLYSLSSVVT QSGNSQESVTEQDSKDSTYSLS VPSSSLGTKTYTCNVDHKPSNTKV STLTLSKADYEKHKVYACEVTH DKRVESKYGPPCPPCPAPEAAGGP QGLSSPVTKSFNRGEC SVFLFPPKPKDTLMISRTPEVTCV VVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK

The interactions of parent PSMA mAbs PSMB123 (Fab PSMB55), PSMB127 (Fab PSMB83), and PSMB130 (Fab PSMB86) with human, chimp, and cyno PSMA ECDs were measured by Surface Plasmon Resonance (SPR) using a ProteOn XPR36 system (BioRad) as described previously for recombinant chimp PSMA ECD. The summary of binding affinities for each of these mabs to human, chimp, and cyno PSMA ECD are shown in Table 18. These mAbs bind targets with similar affinities to the bispecific antibodies.

TABLE 18 Binding of monoclonal antibodies to recombinant human, chimp, and cyno PSMA ECD by Proteon Human KD Chimp KD Cyno KD (nM) (nM) (nM) PSMB123 9.03 ± 1.20 14.3 ± 3.33 66.7 ± 8.60 (Fab PSMB55) PSMB127 12.0 ± 2.05 12.8 ± 1.83 6.68 ± 0.45 (Fab PSMB83) PSMB130 29.6 ± 2.43 31.7 ± 7.48 >300 (Fab PSMB86)

Example 3: Human Framework Adaptation of Anti-CD3 Antibody SP34

Anti-CD3 murine antibody SP34 was humanized by the Human Framework Adaptation method (Fransson, et al, JMB, 2010 398(2):214-31). The VH and VL sequences of SP34 are shown below and in FIG. 4, with residues 1-215 of the light chain and residues 1-230 of the heavy chain derived directly from the electron density map, and with residues 231-455 derived from IGHG3_MOUSE (mouse IgG3, isoform 2).

VH of SP34 (SEQ ID NO: 128) EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWV ARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYY CVRHGNFGNSYVSWFAYWGQGTLVTVSA VL of SP34 (SEQ ID NO: 129) QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLI GGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVF GGGTKLTVL

Four different heavy chains were combined with three different light chains to produce 12 humanized variants.

SP34 Humanization and Affinity Maturation Selection of Human Germlines

A matrix of four human heavy and three light variable region sequences were selected for testing. Selection of human germlines were based solely on the overall sequence similarity to SP34 in the framework region (FR). Neither the CDR sequences, nor their length or canonical structures, were considered in this selection.

The closest matches for the heavy chain are human GLs IGHV3-72 and IGHV3-73. Another GL, IGHV3-23 was selected because of its high frequency of occurrence in the human B-cell repertoire.

The closest matches for the light chain are human lambda GLs IGLV7-43 (aka 7a), IGLV7-46 (aka 7b) and IGLV1-51 (aka 1b). IGLV7-46 is virtually identical to IGLV7-43, but has an advantage of Ala at position 2, i.e. as in SP34.

Selected J-regions are the following: IGHJ1 for the heavy chain; IGLJ3 for the lambda light chain

Back Mutations

To preserve the conformation of CDR-H3, residues in several framework positions in VL, most notably positions Val38, Gly48 and Gly51 must be retained. These ‘back mutations’ were added into the humanization plan.

The Asn at position 57 of the heavy chain was truncated to Gly in the maturation plan to allow necessary flexibility and potentially improve stability (by reducing non-glycine related local structural strain) while not impacting binding.

There were several other considerations made in the humanization design. First, human GLs IGLV7-46 and IGLV7-43 introduce a Trp at position 59 with an unwanted oxidation potential. Two other GLs have Gly at this position, which corresponds to the mouse sequence. Therefore, Gly59 was preserved in both IGLV7-46 and IGLV7-43 variants. Finally, Ala at position 49 of VH may be essential. Also, the residue at position 99 (Val in SP34) may impact antigen binding. To test these positions, back mutations were introduced in some variants (FIG. 5)

HFA Matrix

The HFA matrix (Table 8) is composed of four variants of VH and three variants of VL (FIG. 5). For the purpose of HFA, AbM CDR definition (K. R. Abhinandan and A. C. Martin, 2008. Mol. Immunol. 45, 3832-3839) is used.

The variants for VH:

CD3H141 (SEQ ID NO: 104): EGHV3-72*01 with mouse CDRs + Gly49Ala EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVAR IRSKYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR HGNFGNSYVSWFAYWGQGTLVTVSS CD3H142 (SEQ ID NO: 102): IGHV3-23*01 with mouse CDRs + Ser49Ala EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVAR IRSKYNNYATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK HGNFGNSYVSWFAYWGQGTLVTVSS CD3H143 (SEQ ID NO: 115): IGHV3-23*01 with mouse CDRs + Ser49Ala, Ala99Val EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVAR IRSKYNNYATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVK HGNFGNSYVSWFAYWGQGTLVTVSS CD3H144 (SEQ ID NO: 116): IGHV3-73*01 with mouse CDRs + Asn57Gly EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQASGKGLEWVGR IRSKYNGYATYYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTR HGNFGNSYVSWFAYWGQGTLVTVSS

The variants for VL:

CD3L63 (SEQ ID NO: 103): IGLV7-46*01 with mouse CDRs + Ser49Ala QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLI GGTNKRAPGRPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVF GGGTLKTVL CD3L64 (SEQ ID NO: 117): IGLV1-51*01 with mouse CDRs + Y38V, L48G, Y51G QSVLTQPPSVSAAPGQKVTISCRSSTGAVTTSNYANWVQQLPGTAPKGLI GGTNKRAPGIPDRFSGSKSGTSATLGITGLQTGDEADYYCALWYSNLWVF GGGTLKTVL CD3L66 (SEQ ID NO: 105): IGLV7-43*01 with mouse CDRs + F38V, A48G, Y51G, W59G QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGSLLGGKAALTLSGVQPFDFAEYYCALWYSNLWVF GGGTKLTVL

TABLE 8 Matrix of CD3 Heavy and Light chains (All were prepared with IgG1-AA Fc containing L234A, L235A, and F405L) CD3L63 (LV7- CD3L64 (LV1- CD3L66 (LV7- 46/W59G) SEQ 51) SEQ 43/W59G) SEQ ID NO: 103 ID NO: 117 ID NO: 105 CD3H141 CD3B143 CD3B144 CD3B146 (HV3-72 + G49A) SEQ ID NO: 104 CD3H142 CD3B147 CD3B148 CD3B150 (HV3-23 + S49A) SEQ ID NO: 102 CD3H143 (HV3- CD3B151 CD3B152 CD3B154 23 + S49A, A99V) SEQ ID NO: 115 CD3H144 (VH3- CD3B155 CD3B156 CD3B158 73 with G49) SEQ ID NO: 116

Amino acid sequences were back-translated to DNA and cDNA was prepared using gene synthesis techniques (U.S. Pat. Nos. 6,670,127; 6,521,427). Heavy chain (HC) variable regions were subcloned onto human IgG1-AA Fc containing L234A, L235A, and F405L mutations using an in-house expression vector with the CMV promoter using standard molecular biology techniques. Light chain (LC) variable regions were subcloned onto a human Lambda (λ) constant regions using an in-house expression vector with the CMV promoter using standard molecular biology techniques. Resulting plasmids were transfected into Expi293F cells (Invitrogen) and mAbs were expressed. Purification was by standard methods using a Protein A column (hiTrap MAbSelect SuRe column). After elution, the pools were dialyzed into D-PBS, pH 7.2. The VH and VL sequence of the antibodies are shown in Table 9.

TABLE 9 The VH and VL sequences of anti-CD3 antibodies SEQ SEQ VH Amino Acid ID VL Amino Acid ID mAb HC sequence NO: LC sequence NO: CD3B1 CD3H EVQLVESGGGLVQPG 104 CD3L QAVVTQEPSLTVSP 103 43 141 GSLRLSCAASGFTFN 63 GGTVTLTCRSSTGA TYAMNWVRQAPGKGL VTTSNYANWVQQKP EWVARIRSKYNNYAT GQAPRGLIGGTNKR YYAASVKGRFTISRD APGTPARFSGSLLG DSKNSLYLQMNSLKT GKAALTLSGAQPED EDTAVYYCARHGNFG EAEYYCALWYSNLW NSYVSWFAYWGQGTL VFGGGTKLTVL VTVSS CD3B1 CD3H EVQLVESGGGLVQPG 104 CD3L QSVLTQPPSVSAAP 117 44 141 GSLRLSCAASGFTFN 64 GQKVTISCRSSTGA TYAMNWVRQAPGKGL VTTSNYANWVQQLP EWVARIRSKYNNYAT GTAPKGLIGGTNKR YYAASVKGRFTISRD APGIPDRFSGSKSG DSKNSLYLQMNSLKT TSATLGITGLQTGD EDTAVYYCARHGNFG EADYYCALWYSNLW NSYVSWFAYWGQGTL VFGGGTKLTVL VTVSS CD3B1 CD3H EVQLVESGGGLVQPG 104 CD3L QTVVTQEPSLTVSP 105 46 141 GSLRLSCAASGFTFN 66 GGTVTLTCRSSTGA TYAMNWVRQAPGKGL VTTSNYANWVQQKP EWVARIRSKYNNYAT GQAPRGLIGGTNKR YYAASVKGRFTISRD APGTPARFSGSLLG DSKNSLYLQMNSLKT GKAALTLSGVQPED EDTAVYYCARHGNFG EAEYYCALWYSNLW NSYVSWFAYWGQGTL VFGGGTKLTVL VTVSS CD3B1 CD3H EVQLLESGGGLVQPG 102 CD3L QAVVTQEPSLTVSP 103 47 142 GSLRLSCAASGFTFN 63 GGTVTLTCRSSTGA TYAMNWVRQAPGKGL VTTSNYANWVQQKP EWVARIRSKYNNYAT GQAPRGLIGGTNKR YYADSVKGRFTISRD APGTPARFSGSLLG NSKNTLYLQMNSLRA GKAALTLSGAQPED EDTAVYYCAKHGNFG EAEYYGALWYSNLW NSYVSWFAYWGQGTL VFGGGTKLTVL VTVSS CD3B1 CD3H EVQLLESGGGLVQPG 102 CD3L QSVLTQPPSVSAAP 117 48 142 GSLRLSCAASGFTFN 64 GQKVTISCRSSTGA TYAMNWVRQAPGKGL VTTSNYANWVQQLP EWVARIRSKYNNYAT GTAPKGLIGGTNKR YYADSVKGRFTISRD APGIPDRFSGSKSG NSKNTLYLQMNSLRA TSATLGITGLQTGD EDTAVYYCAKHGNFG EADYYCALWYSNLW NSYVSWFAYWGQGTL VFGGGTKLTVL VTVSS CD3B1 CD3H EVQLLESGGGLVQPG 102 CD3L QTVVTQEPSLTVSP 105 50 142 GSLRLSCAASGFTFN 66 GGTVTLTCRSSTGA TYAMNWVRQAPGKGL VTTSNYANWVQQKP EWVARIRSKYNNYAT GQAPRGLIGGTNKR YYADSVKGRFTISRD APGTPARFSGSLLG NSKNTLYLQMNSLRA GKAALTLSGVQPED EDTAVYYCAKHGNFG EAEYYCALWYSNLW NSYVSWFAYWGQGTL VFGGGTKLTVL VTVSS CD3B1 CD3H EVQLVESGGGLVQPG 115 CD3L QAVVTQEPSLTVSP 103 51 143 GSLRLSCAASGFTFN 63 GGTVTLTCRSSTGA TYAMNWVRQAPGKGL VTTSNYANWVQQKP EWVARIRSKYNNYAT GQAPRGLIGGTNKR YYADSVKGRFTISRD APGTPARFSGSLLG NSKNTLYLQMNSLRA GKAALTLSGAQPED EDTAVYYCVKHGNFG EAEYYCALWYSNLW NSYVSWFAYWGQGTL VFGGGTKLTVL VTVSS CD3B1 CD3H EVQLLESGGGLVQPG 115 CD3L QSVLTQPPSVSAAP 117 52 143 GSLRLSCAASGFTFN 64 GQKVTISCRSSTGA TYAMNWVRQAPGKGL VTTSNYANWVQQLP EWVARIRSKYNNYAT GTAPKGLIGGTNKR YYADSVKGRFTISRD APGIPDRFSGSKSG NSKNTLYLQMNSLRA TSATLGITGLQTGD EDTAVYYCVKHGNFG EADYYCALWYSNLW NSYVSWFAYWGQGTL VFGGGTKLTVL VTVSS CD3B1 CD3H EVQLLESGGGLVQPG 115 CD3L QTVVTQEPSLTVSP 105 54 143 GSLRLSCAASGFTFN 66 GGTVTLTCRSSTGA TYAMNWVRQAPGKGL VTTSNYANWVQQKP EWVARIRSKYNNYAT GQAPRGLIGGTNKR YYADSVKGRFTISRD APGTPARFSGSLLG NSKNTLYLQMNSLRA GKAALTLSGVQPED EDTAVYYCVKHGNFG EAEYYCALWYSNLW NSYVSWFAYWGQGTL VFGGGTKLTVL VTVSS CD3B1 CD3H EVQLLESGGGLVQPG 116 CD3L QAVVTQEPSLTVSP 103 55 144 GSLKLSCAASGFTFN 63 GGTVTLTCRSSTGA TYAMNWVRQASGKGL VTTSNYANWVQQKP EWVGRIRSKYNGYAT GQAPRGLIGGTNKR YYAASVKGRFTISRD APGTPARFSGSLLG DSKNTAYLQMNSLKT GKAALTLSGAQPED EDTAVYYCTRHGNFG EAEYYCALWYSNLW NSYVSWFAYWGQGTL VFGGGTKLTVL VTVSS CD3B1 CD3H EVQLVESGGGLVQPG 116 CD3L QSVLTQPPSVSAAP 117 56 144 GSLKLSCAASGFTFN 64 GQKVTISCRSSTGA TYAMNWVRQASGKGL VTTSNYANWVQQLP EWVGRIRSKYNGYAT GTAPKGLIGGTNKR YYAASVKGRFTISRD APGIPDRFSGSKSG DSKNTAYLQMNSLKT TSATLGITGLQTGD EDTAVYYCTRHGNFG EADYYCALWYSNLW NSYVSWFAYWGQGTL VFGGGTKLTVL VTVSS CD3B1 CD3H EVQLVESGGGLVQPG 116 CD3L QTVVTQEPSLTVSP 105 58 144 GSLKLSCAASGFTFN 66 GGTVTLTCRSSTGA TYAMNWVRQASGKGL VTTSNYANWVQQKP EWVGRIRSKYNGYAT GQAPRGLIGGTNKR YYAASVKGRFTISRD APGTPARFSGSLLG DSKNTAYLQMNSLKT GKAALTLSGVQPED EDTAVYYCTRHGNFG EAEYYCALWYSNLW NSYVSWFAYWGQGTL VFGGGTKLTVL VTVSS

Example 4: Endogenous Cell Binding of the Humanized Anti-CD3 Hits to Primary T Cells

The resulting panel of anti-CD3 antibodies was tested for binding against cell-surface CD3ε on primary human T cells. To do this, binding of antibodies from expression supernatants was visualized using a polyclonal anti-human secondary antibody and analyzed by flow cytometry. Briefly, binding of anti-CD3 antibodies to cell-surface CD3E was assessed by flow cytometry using primary Human T lymphocytes purified by negative selection (Biological Specialty, Colmar, USA). Expression supernatants or purified antibodies were normalized to 10 μg/ml in media or FACS buffer (BD BioSciences), respectively. 2×10⁵ cells were aliquoted into wells of a 96 well round-bottomed plate (CoStar) for labeling. Antibodies in expression supernatant were added to cells and incubated for 45 min at 4° C. Following centrifugation at 1300 rpm for 3 min and removal of supernatant, 50 μL of anti-human IgG (H+L) Alexa Fluor 647 secondary antibody (Life technologies Inc.) was incubated with the cells at a final concentration of 10 μg/mL for 30 min at 4° C. away from direct light. Following washing and resuspension in 30 μL FACs buffer (BD BioSciences). Sample collection was performed on an Intellicyt HTFC system using ForeCyt software. Viable single cells were gated prior to analysis of binding using the green or red fixable live/dead dyes (Life Technologies Inc.) and forward/side scatter area and height parameters, respectively. Graphs were generated in GraphPad Prism version 5 using mean fluorescence intensity values.

Although a titration series was run, an intermediate concentration is presented in FIG. 6 for clarity. Two in-house phage-derived antibodies with the same Fc region as the therapeutic antibodies were used as controls: GI (HC SEQ ID NO: 118, LC SEQ ID NO: 119), a non-cyno cross-reactive, agonistic antibody was used as a positive control and CD3B94 (HC-SEQ ID NO: 120, LC-SEQ ID NO: 121) a non-binder/non-agonistic antibody was used to assess non-specific binding. The commercial SP34 antibody was not used as a comparator in this assay since it is a mouse antibody and the use of a different secondary detection reagent would have prohibited direct comparison with the variants tested.

The data demonstrates an array of binding potential within the panel of humanized anti-CD3 hits, with two antibodies (CD3B144, CD3B152) showing complete loss of binding to human T cells The remaining antibodies showed a range of binding potential that could be broadly split into strong and weak binders using G11 binding as an arbitrary threshold. Using these parameters, seven strong binders and seven weak binders were identified from the panel of variants (FIG. 6).

Binding analysis of the anti-CD3 hits to primary cynomolgus CD4⁺ T cells was then tested in order to assess the retention of cross-reactivity. Purified CD4⁺ T cells from the peripheral blood of cynomolgus monkeys (Zen Bio, Triangle Research Park, USA were used). Assay protocols were similar to those described above. Since G11 does not cross-react with cynomolgus CD3ε, CD3B124, an in-house chimeric SP34-derived antibody having the VH and VL of SP34 with murine framework and a human IgG1 Fc was used as a positive control in this assay (FIG. 7). Interestingly, several variants showed decreased binding potential compared to that seen with human cells. This included the strong binders CD3B50, CD3B151 and CD3B154, in which binding was reduced, and several weak binders where binding could no longer be detected over background. This loss of binding was not related to a specific immunoglobulin chain, suggesting that the combination of heavy and light chains played a role in the loss of cross-reactivity. Together, these assays allowed the identification of variants that retained species cross-reactivity between human and cynomolgus CD3ε.

Example 5: Functional Analysis of the Humanized Anti-CD3 Hits in Primary T Cells

Binding analysis demonstrated that the panel of humanized anti-CD3 hits showed a range of binding potential to human and cynomolgus T-cells. To investigate the capacity of each variant to induce activation via CD3ε crosslinking, primary T-cells were cultured overnight in the presence of bead-conjugated antibody. The following day, cells were harvested and labeled with an anti-CD69 antibody to measure activation (FIG. 8). Humanized anti-CD3 antibodies were bound to protein A coated magnetic beads (SpheroTech, Lake forest, USA) by overnight incubation with antibody at 10 μg/mL. The following day, 2×10⁵ primary human T cells were plated in round-bottomed cell culture plates in triplicate and 2×10⁵ coated beads were added. Following overnight culture at 37° C., cells were harvested and labeled with anti-CD69 Alexa Fluor® 488 antibody (clone FN50; Biolegend) to assess the up-regulation of this activation marker. Sample collection and analysis were performed as described above for binding. Several negative controls were run, including T-cells alone, T-cells with non-coated beads, and T-cells with isotype control (CD3B94)-coated beads. All of these showed similar mean fluorescence intensity values comparable to unstained T-cells indicating that background was low in this assay. Several positive controls were run for comparison, including OKT3 (U.S. Pat. No. 5,929,212) and commercially available SP34-2 antibody.

The humanized anti-CD3 hits were then tested for their capacity to activate primary cynomolgus CD4+ T cells (Zen Bio, Triangle Research Park, USA) in the same assay (FIG. 9). The FN50 anti-CD69 antibody has been described as being cross-reactive with non-human protein and could therefore be used to test activation of these cells.

The human and cynomolgus activation data correlated with the binding data in that the panel of hits displayed a range of activation potentials. A number of the strong binders showed the capacity to activate human T-cells to an equivalent or greater extent when compared to commercially available SP34-2. Several variants showed activation potential that was lower compared SP34-2, whereas some binders did not show evidence of CD69 stimulation. The inability to activate was only seen in the variants that showed no or weak binding and all strong binders showed some level of activation, suggesting a correlation between binding and activation potentials for both human (FIG. 10A) and cynomolgus (FIG. 10B).

Two anti-CD3 antibodies, CD3B146 and CD3B147, with high and medium affinity respectively, were selected for preparation of bispecific antibodies with the PSMA specific antibodies. These two anti-CD3 antibodies were prepared in the IgG4 PAA GenMab format (Labrijn et al, 2013) where the targeting parent (PSMA) contains the 409R GenMab mutation (native amino acid for IgG4), while the killing parent (CD3) contains the F405L GenMab mutation and R409K. The monospecific anti-CD3 antibody was expressed as IgG4, having Fc substitutions S228P, F234A, L235A, F405L, and R409K (CD3 arm) (numbering according to EU index) in their Fc regions. Heavy chain (HC) variable regions were subcloned onto human IgG4-PAA Fc containing S228P, F234A, L235A, F405L, and R409K mutations using an in-house expression vector with the CMV promoter using standard molecular biology techniques. Light chain (LC) variable regions were subcloned onto a human Lambda (λ) constant regions using an in-house expression vector with the CMV promoter using standard molecular biology techniques. Resulting plasmids were transfected into Expi293F cells (Invitrogen) and mAbs were expressed. The anti-CD3 antibodies were purified using standard purification methods: a protein A column with an elution buffer of 100 mM NaAc pH3.5 and a neutralization puffer of 2M Tris pH 7.5 and 150 mM NaCl. The mabs were desalted using PD10 (Sephadex G25M) column and the pools were dialyzed into D-PBS, pH 7.2.

A monospecific anti-CD3 antibody CD3B217 was generated comprising the VH and VL regions having the VH of SEQ ID NO:102 and the VL of SEQ ID NO:103 and an IgG4 constant region with S228P, F234A, L235A, F405L, and R409K substitutions. CD3B217 comprises a heavy chain of SEQ ID NO: 108 and a light chain of SEQ ID NO:109. A monospecific anti-CD3 antibody CD3B219 was generated comprising the VH and VL regions having the VH of SEQ ID NO:104 and the VL of SEQ ID NO:105 and an IgG4 constant region with S228P, F234A, L235A, F405L, and R409K substitutions. CD3B219 comprises a heavy chain of SEQ ID NO: 110 and a light chain of SEQ ID NO:111.

Example 6. Preparation of PSMA×CD3 Bispecific Antibodies

The formation of the PSMA×CD3 Bispecific antibodies requires two parental mAbs, one specific for the targeting arm (e.g. PSMA) and one specific for the effector arm (e.g. CD3). PSMA mAbs were recombined with a medium affinity CD3B217 (VH SEQ ID NO: 102, VL SEQ ID NO:103) and a high affinity CD3B219 (VH SEQ ID NO:104, VL SEQ ID NO:105) CD3 arms. These parental mAbs are in the GenMab format (Labrijn et al, 2013) where the targeting parent (PSMA) contains the 409R GenMab mutation (native amino acid for IgG4), while the killing parent (CD3) contains the F405L GenMab mutation and R409K mutation. The monospecific anti-CD3 antibody was expressed as IgG4, having Fc substitutions S228P, F234A, L235A, F405L, and R409K (CD3 arm) (numbering according to EU index) in their Fc regions. The monospecific antibodies were expressed in HEK cell lines under CMV promoters.

The parental PSMA and CD3 antibodies were purified using a protein A column with an elution buffer of 100 mM NaAc pH3.5 and a neutralization puffer of 2M Tris pH 7.5 and 150 mM NaCl. The mabs were desalted using PD10 (Sephadex G25M) column and dialyzed intoD-PBS, pH 7.2 buffer.

Post purification the parental PSMA antibodies were mixed with the desired parental CD3 antibody under reducing conditions in 75 mM cysteamine-HC1 and incubated at 31° C. for 4h. The recombination reactions were based on molar ratios, where a set amount of PSMA (eg, 10 mg, or ˜67.8 nanomoles) was combined with CD3 antibody (eg, ˜71.8 nanomoles), where the CD3 antibody was added in a 6% excess of the PSMA antibody. The concentrations of the PSMA antibody stocks varied from 0.8 to 6 mg/mL, and the volumes the recombination reactions varied for each pairing. The recombinations were subsequently dialyzed against PBS to remove the reductant. The bispecific antibody reactions were performed with an excess of the CD3 antibody (ratio) to minimize the amount of unreacted PSMA parental antibody remaining after recombination. Following the partial reduction of the parental mAbs, the reductant was removed via overnight dialysis into PBS.

The final bispecific antibodies produced, along with the parental mAbs (i.e. PSMA, CD3, or Null) used in the recombination reactions are listed in Table 10, with sequences listed in Tables 11 and 12.

Selected PSMA hits were also paired with a non-killing arm (Null) to create negative controls for testing purposes. For control bispecific antibodies, B2M1, an RSV antibody in the IgG4 PAA format (VH SEQ ID NO: 106, VL SEQ ID NO: 107) was generated, purified and, combined with either the CD3 arm CD3B219-F405L, R409K to generate CD3B288 (CD3 X null) or PSMA arms, PSMB162, PSMB126, PSMB130 to generate PS3B37, PS3B39 and PS3B40 respectively (PSMA X null).

TABLE 10 PSMA × CD3 Bispecific antibodies generated SEQ ID NO: (VH; VL IgG4PAA F405L, Bispecific SEQ ID NO: R409K) antibody ARM 1 HC LC ARM 2 HC LC PS3B2 PSMB120 99 100 CD3B217 108 109 (FAB PSMB25) PS3B3 PSMB121 97 98 CD3B217 108 109 (FAB PSMB49) PS3B4 PSMB122 96 83 CD3B217 108 109 (FAB PSMB51) PS3B5 PSMB123 94 95 CD3B217 108 109 (FAB PSMB55) PS3B7 PSMB87 125 91 CD3B217 108 109 (FAB PSMB58) PS3B8 PSMB126 90 91 CD3B217 108 109 (FAB PSMB80) PS3B9 PSMB127 88 89 CD3B217 108 109 (FAB PSMB83) PS3B10 PSMB128 86 87 CD3B217 108 109 (FAB PSMB84) PS3B11 PSMB129 82 83 CD3B217 108 109 (FAB PSMB109) PS3B12 PSMB130 84 85 CD3B217 108 109 (FAB PSMB86) PS3B19 PSMB119 101 100 CD3B219 110 111 (FAB PSMB18) PS3B20 PSMB120 99 100 CD3B219 110 111 (FAB PSMB25) PS3B21 PSMB121 97 98 CD3B219 110 111 (FAB PSMB49) PS3B22 PSMB122 96 83 CD3B219 110 111 (FAB PSMB51) PS3B23 PSMB123 94 95 CD3B219 110 111 (FAB PSMB55) PS3B24 PSMB124 92 93 CD3B219 110 111 (FAB PSMB56) PS3B25 PSMB87 125 91 CD3B219 110 111 (FAB PSMB58) PS3B26 PSMB126 90 91 CD3B219 110 111 (FAB PSMB80) PS3B27 PSMB127 88 89 CD3B219 110 111 (FAB PSMB83) PS3B28 PSMB128 86 87 CD3B219 110 111 (FAB PSMB84) PS3B29 PSMB129 82 83 CD3B219 110 111 (FAB PSMB109) PS3B30 PSMB130 84 85 CD3B219 110 111 (FAB PSMB86)

A monospecific anti-CD3 antibody CD3B217 was generated comprising the VH and VL regions having the VH of SEQ ID NO:102 and the VL of SEQ ID NO:103 and an IgG4 constant region with S228P, F234A, L235A, F405L, and R409K substitutions. CD3B217 comprises a heavy chain of SEQ ID NO: 108 and a light chain of SEQ ID NO:109. A monospecific anti-CD3 antibody CD3B219 was generated comprising the VH and VL regions having the VH of SEQ ID NO:104 and the VL of SEQ ID NO:105 and an IgG4 constant region with S228P, F234A, L235A, F405L, and R409K substitutions. CD3B219 comprises a heavy chain of SEQ ID NO: 110 and a light chain of SEQ ID NO: 111. As a control, a monospecific anti-RSV antibody, derived from B21M, was generated comprising the VH and VL regions having the VH of SEQ ID NO: 106 and the VL of SEQ ID NO:107 and an IgG4 constant region with S228P, F234A, L235A, or F234A, L235A, R409K, F405L to partner as the null arm with either the CD3 or PSMA arm of a bispecific antibody.

The bispecific antibody PS3B2 comprises the CD3 binding arm of mAb CD3B217-F405L, R409K and the PSMA binding arm of mAb PSMB120. The bispecific antibody PS3B3 comprises the CD3 binding arm of mAb CD3B217-F405L, R409K and the PSMA binding arm of mAb PSMB121-R409. The bispecific antibody PS3B4 comprises the CD3 binding arm of mAb CD3B217-F405L, R409K and the PSMA binding arm of mAb PSMB122-R409. The bispecific antibody PS3B5 comprises the CD3 binding arm of mAb CD3B217-F405L, R409K and the PSMA binding arm of mAb PSMB123-R409. The bispecific antibody PS3B7 comprises the CD3 binding arm of mAb CD3B217-F405L, R409K and the PSMA binding arm of mAb PSMB58-R409. The bispecific antibody PS3B8 comprises the CD3 binding arm of mAb CD3B217-F405L, R409K and the PSMA binding arm of mAb PSMB126-R409. The bispecific antibody PS3B9 comprises the CD3 binding arm of mAb CD3B217-F405L, R409K and the PSMA binding arm of mAb PSMB127-R409. The bispecific antibody PS3B10 comprises the CD3 binding arm of mAb CD3B217-F405L, R409K and the PSMA binding arm of mAb PSMB128-R409. The bispecific antibody PS3BI 1 comprises the CD3 binding arm of CD3B217-F405L, R409K and the PSMA binding arm of mAb PSMB129-R409. The bispecific antibody PS3B12 comprises the CD3 binding arm of CD3B217-F405L, R409K and the PSMA binding arm of mAb PSMB130-R409.

The bispecific antibody PS3B19 comprises the CD3 binding arm of mAb CD3B219-F405L, R409K and the PSMA binding arm of mAb PSMB119-R409. The bispecific antibody PS3B20 comprises the CD3 binding arm of mAb CD3B219-F405L, R409K and the PSMA binding arm of mAb PSMB120-R409. The bispecific antibody PS3B21 comprises the CD3 binding arm of mAb CD3B219-F405L, R409K and the PSMA binding arm of mAb PSMB121-R409. The bispecific antibody PS3B22 comprises the CD3 binding arm of mAb CD3B219-F405L, R409K and the PSMA binding arm of mAb PSMB122-R409. The bispecific antibody PS3B23 comprises the CD3 binding arm of mAb CD3B219-F405L, R409K and the PSMA binding arm of mAb PSMB123-R409. The bispecific antibody PS3B24 comprises the CD3 binding arm of mAb CD3B219-F405L, R409K and the PSMA binding arm of mAb PSMB124-R409. The bispecific antibody PS3B25 comprises the CD3 binding arm of mAb CD3B219-F405L, R409K and the PSMA binding arm of mAb PSMB165-R409. The bispecific antibody PS3B26 comprises the CD3 binding arm of mAb CD3B219-F405L, R409K and the PSMA binding arm of mAb PSMB126-R409. The bispecific antibody PS3B27 comprises the CD3 binding arm of CD3B219-F405L, R409K and the PSMA binding arm of mAb PSMB127-R409. The bispecific antibody PS3B28 comprises the CD3 binding arm of CD3B219-F405L, R409K and the PSMA binding arm of mAb PSMB128-R409. The bispecific antibody PS3B29 comprises the CD3 binding arm of CD3B219-F405L, R409K and the PSMA binding arm of mAb PSMB129-R409. The bispecific antibody PS3B30 comprises the CD3 binding arm of CD3B219-F405L, R409K and the PSMA binding arm of mAb PSMB130-R409.

TABLE 11 Sequences of PSMA X CD3 bispecific antibodies Heavy Chain Amino Light Chain Amino  acid sequences: SEQ Acid Sequences: SEQ Bispccific PSMA Arm ID PSMA Arm ID Ab ID CD3 Arm NO CD3 Arm NO PS3B2 EVQLVQSGAEVKKPGESLKISCK  99 DIVMTOSPDSLAVSLGERATI 100 (PSMB120 GSGYSFTSYWIGWVRQMPGKGL NCKSSQSVINSSNNKNYLA X EWMGIIYPGDSDTRYSPSFQGQVT WYQQKPGQPPKILIYWAST CD3B217) ISADKSISTAYLQWSSLKASDTAM RESGVPDRFSGSGSGTDFTL YYCARGWAYDRGLDYWGQGTL TISSLOAEDVAVYYCQQYYS VTVSSASTKGPSVFPLAPCSRSTSE TPLTFGQGTKVEIKRTVAAP STAALGCLVKDYFPEPVTVSWNS SVFIFPPSDEQLKSGTASVVC GALTSGVHTFPAVLQSSGLYSLSS LLNNFYPREAKVQWKVDNA VVTVPSSSLGTKTYTCNVDHKPS LQSGNSQESVTEQDSKDSTY NTKVDKRVESKYGPPCPPCPAPE SLSSTLTLSKADYEKHKVYA AAGGPSVFLIPPKPKDTLMISRTP CEVTHQGLSSPVTKSFNRGE EVTCVVVDVSQEDPEVQFNWYV C DGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVS NKGLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQK SLSLSLGK EVQLLESGGGLVQPGGSLRLSCA 108 QAVVTQEPSLTVSPGGTVTL 109 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYADSVK QKPGQAPRGLIGGTNKRAPG GRFTISRDNSKNTLYLQMNSLRA TPARFSGSLLGGKAATLTLSG EDTAVYYCVKHGNFGNSYVSWF AQPEDEAEYYCALWYSNLW AYWGQGTLVTVSSASTKGPSVFP VFGGGTKLTVLGQPKAAPS LAPCSRSTSESTAALGCLVKDYFP VTLFPPSSEELQANKATLVC EPVTVSWNSGALTSGVHTFPAVL LISDFYPGAVTVAWKADSSP QSSGLYSLSSVVTVPSSSLGTKTY VKAGVETTTPSKQSNNKYA TCNVDHKPSNTKVDKRVESKYGP ASSYLSLTPEQWKSHRSYSC PCPPCPAPEAAGGPSVFLFPPKPK QVTHEGSTVEKTVAPTECS DTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B3 QVQLVQSGAEVKKPGSSVKVSCK  97 EIVLTQSPATLSLSPGERATL 98 (PSMB121 ASGGTFSSYAISWVRQAPGQGLE SCRASQSVSKWLAWYQQKP X WMGGIIPIFGTANYAQKFQGRVTI GQAPRLLIYDASNRATGIPA CD3B217) TADESTSTAYMELSSLRSEDTAV RFSGSGSGTDFTLTISSLEPE YYCARASRVWHASYGYLDYWG DFAVYYCQQRFTAPWTFGQ QGTLVTVSSASTKGPSVFPLAPCS GTKVEIKRTVAAPSVFIFPPS RSTSESTAALGCLVKDYFPEPVTV DEQLKSGTASVVCLLNNFYP SWNSGALTSGVHTFPAVLQSSGL REAKVQWKVDNALQSGNS YSLSSVVTVPSSSLGTKTYTCNVD QESVTEQDSKDSTYSLSSTLT HKPSNTKVDKRVESKYGPPCPPC LSKADYEKHKVYACEVTHQ PAPEAAGGPSVFLFPPKPKDTLMI GLSSPVTKSENRGEC SRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNS TYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK EVQLLESGGGLVQPGGSLRLSCA 108 QAVVTQFPSLTVSPGGTVTL 109 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYADSVK QKPGQAPRGLIGGTNKRAPG GRFTISRDNSKNTLYLQMNSLRA TPARFSGSLLGGKAALTLSG EDTAVYYCVKHGNFGNSYVSWF AQPEDEAEYYCALWYSNLW AYWGQGTLVTVSSASTKGPSVFP VFGGGTKLTVLGQPKAAPS LAPCSRSTSESTAALGCLVKDYFP VTLFPPSSEELQANKATLVC EPVTVSWNSGALTSGVHTFPAVL LISDFYPGAVTVAWKADSSP QSSGLYSLSSVVTVPSSSLGTKTY VKAGVETTTPSKQSNNKYA TCNVDHKPSNTKVDKRVESKYGP ASSYLSLTPEQWKSHRSYSC PCPPCPAPEAAGGPSVFLFPPKPK QVTHEGSTVEKTVAPTECS DTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B4 QVQLVQSGAEVKKPGSSVKVSCK  96 DIQMTQSPSSLSASVGDRVTI  83 (PSMB122 ASGGTFSSYAISWVRQAPGQGLE TCRASQSIDRWLNWYQQKP X WMGWIIPYNGNANYAQKFQGRV GKAPKLLIYAASSLQSGVPS CD3B217) TITADESTSTAYMELSSLRSEDTA RFSGSGSGTDFTLTISSLQPE VYYCARVNSAALVWERLDYWG DFATYYCQQSPRYPLTFGQG QGTLVTVSSASTKGPSVFPLAPCS TKVEIKRTVAAPSVFIFPPSD RSTSESTAALGCLVKDYFPEPVTV EQLKSGTASVVCLLNNFYPR SWNSGALTSGVHTFPAVLQSSGL EAKVQWKVDNALQSGNSQE YSLSSVVTVPSSSLGTKTYTCNVD SVTEQDSKDSTYSLSSTLTLS HKPSNTKVDKRVESKYGPPCPPC KADYEKHKVYACEVTHQGL PAPEAAGGPSVFLFPPKPKDTLMI SSPVTKSFNRGEC SRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNS TYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVESCSVMHEALHNHY TQKSLSLSLGK EVQLLESGGGLVQPGGSLRLSCA 108 QAVVTQEPSLTVSPGGTVTL 109 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYADSVK QKPGQAPRGLIGGINKRAPG GRFTISRDNSKNTLYLQMNSLRA TPARFSGSLLGGKAALTLSG EDTAVYYCVKHGNFGNSYVSWF AQPEDEAEYYCALWYSNLW AYWGQGTLVTVSSASTKGPSVFP VFGGGTKLTVLGQPKAAPS LAPCSRSTSESTAALGCLVKDYFP VTLFPPSSEELQANKATLVC EPVTVSWNSGALTSGVHTFPAVL LISDFYPGAVTVAWKADSSP QSSGLYSLSSVVTVPSSSLGTKTY VKAGVETTTPSKQSNNKYA TCNVDHKPSNTKVDKRVESKYGP ASSYLSLTPEQWKSHRSYSC PCPPCPAPEAAGGPSVFLFPPKPK QVTHEGSTVEKTVAPTECS DTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B5 EVQLVQSGAEVKKPGESLKISCK  94 EIVLTQSPATLSLSPGERATL  95 (PSMB123 GSGYSFTSYWIGWVRQMPGKGL SCRASQSVASDLAWYQQKP X EWMGIIYPGDSDTRYSPSFQGQVT GQAPRLLIYFASNRATGIPAR CD3B217) ISADKSISTAYLQWSSLKASDTAM FSGSGSGTDFTLTISSLEPEDF YYCARGLPIWYLDYWGQGTLVT AVYYCQQSITWPFTFGQGTK VSSASTKGPSVFPLAPCSRSTSEST VEIKRTVAAPSVFIFPPSDEQ AALGCLVKDYFPEPVTVSWNSGA LKSGTASVVCLLNNFYPREA LTSGVHTFPAVLQSSGLYSLSSVV KVQWKVDNALQSGNSQESV TVPSSSLGTKTYTCNVDHKPSNT TEQDSKDSTYSLSSTLTLSKA KVDKRVESKYGPPCPPCPAPEAA DYEKHKVYACEVTHQGLSS GGPSVFLFPPKPKDTLMISRTPEV PVTKSFNRGEC TCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSL SLGK EVQLLESGGGLVQPGGSLRLSCA 108 QAVVTQEPSLTVSPGGTVTL 109 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYADSVK QKPGQAPRGLIGGINKRAPG GRFTISRDNSKNTLYLQMNSLRA TPARFSGSLLGGKAALTLSG EDTAVYYCVKHGNFGNSYVSWF AQPEDEAEYYCALWYSNLW AYWGQGTLVTVSSASTKGPSVFP VFGGGTKLTVLGQPKAAPS LAPCSRSTSESTAALGCLVKDYFP VTLFPPSSEELQANKATLVC EPVTVSWNSGALTSGVHTFPAVL LISDFYPGAVTVAWKADSSP QSSGLYSLSSVVTVPSSSLGTKTY VKAGVETTTPSKQSNNKYA TCNVDHKPSNTKVDKRVESKYGP ASSYLSLTPEQWKSHRSYSC PCPPCPAPEAAGGPSVFLFPPKPK QVTHEGSTVEKTVAPTECS DTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B EVQLLESGGGLVQPGGSLRLSCA 108 QAVVTQEPSLTVSPGGTVTL 109 (PSMB87 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ X EWVARIRSKYNNYATYYADSVK QKPGQAPRGLIGGINKRAPG CD3B217) GRFTISRDNSKNTLYLQMNSLRA TPARFSGSLLGGKAALTLSG EDTAVYYCVKHGNFGNSYVSWF AQPEDEAEYYCALWYSNLW AYWGQGTLVTVSSASTKGPSVFP VFGGGTKLTVLGQPKAAPS LAPCSRSTSESTAALGCLVKDYFP VTLFPPSSEELQANKATLVC EPVTVSWNSGALTSGVHTFPAVL LISDFYPGAVTVAWKADSSP QSSGLYSLSSVVTVPSSSLGTKTY VKAGVETTTPSKQSNNKYA TCNVDHKPSNTKVDKRVESKYGP ASSYLSLTPEQWKSHRSYSC PCPPCPAPEAAGGPSVFLFPPKPK QVTHEGSTVEKTVAPTECS DTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK EVQLVQSGAEVKKPGESLKISCK 125 DIQMTQSPSSLSASVGDRVT1  91 GSGYSFTSYWISWVRQMPGKGLE TCRASQSISSYLNWYQQKPG WMGIIYPGDSYTRYSTSFQGQVTI KAPKLLIYAASSLQSGVPSRF SADKSISTAYLQWSSLKASDTAM SGSGSGTDFTLTISSLQPEDF YYCARDYEWELFDSRLDYWGQG ATYYCQQSYSTPLTFGQGTK TLVTVSSASTKGPSVFPLAPCSRS VEIKRTVAAPSVFIFPPSDEQ TSESTAALGCLVKDYFPEPVTVS LKSGTASVVCLLNNFYPREA WNSGALTSGVHTFPAVLQSSGLY KVQWKVDNALQSGNSQESV SLSSVVTVPSSSLGTKTYTCNVDH TEQDSKDSTYSLSSTLTLSKA KPSNTKVDKRVESKYGPPCPPCP DYEKHKVYACEVTHQGLSS APEAAGGPSVFLFPPKPKDTLMIS PVTKSFNRGEC RTPEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK PS3B8 QVQLVQSGAEVKKPGSSVKVSCK  90 DIQMTQSPSSLSASVGDRVTI  91 (PSMB126 ASGGTFDDYAISWVRQAPGQGLE TCRASQSISSYLNWYQQKPG X WMGRIDPIEGTANYAQKFQGRVT KAPKLLIYAASSLQSGVPSRF CD3B217) ITADESTSTAYMELSSERSEDTAV SGSGSGTDFTLTLSSLQPEDF YYCARDRYYYDGVYWYSDYFD ATYYCQQSYSTPLTFGQGTK YWGQGTLVTVSSASTKGPSCFPL VEIKRTVAAPSVFIFPPSDEQ APCSRSTSESTAALGCLVKDYFPE LKSGTASVVCLLNNFYPREA PVTVSWNSGALTSGVHTFPAVLQ KVQWKVDNALQSGNSQESV SSGLYSLSSVVTVPSSSLGTKTYT TEQDSKDSTYSLSSTLTLSKA CNVDHKPSNTKVDKRVESKYGPP DYEKHKVYACEVTHQGLSS CPPCPAPEAAGGPSVFLFPPKPKD PVTKSFNRGEC TLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWENGK EYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK EVQLLESGGGLVQPGGSLRLSCA 108 QAVVTQEPSLTVSPGGTVTL 109 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYADSVK QKPGQAPRGLIGGINKRAPG GRFTISRDNSKNTLYLQMNSLRA TPARFSGSLLGGKAALTLSG EDTAVYYCVKHGNFGNSYVSWF AQPEDEAEYYCALWYSNLW AYWGQGTLVTVSSASTKGPSVFP VFGGGTKLTVLGQPKAAPS LAPCSRSTSESTAALGCLVKDYFP VTLFPPSSEELQANKATLVC EPVTVSWNSGALTSGVHTFPAVL LISDFYPGAVTVAWKADSSP QSSGLYSLSSVVTVPSSSLGTKTY VKAGVETTTPSKQSNNKYA TCNVDHKPSNTKVDKRVESKYGP ASSYLSLTPEQWKSHRSYSC PCPPCPAPEAAGGPSVFLFPPKPK QVTHEGSTVEKTVAPTECS DTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B9 EVQLLESGGGLVQPGGSLRLSCA  88 EIVLTQSPATLSLSPGERATL  89 (PSMB127 ASGFTFKSDAMHWVRQAPGKGL SCRASQSVSSYLAWYQQKP X EWVSEISGSGGYTNYADSVKGRF GQAPRLLIYDASNRATGIPA CD3B217) TISRDNSKNTLYLQMNSLRAEDT RFSGSGSGTDFTLTISSEEPE AVYCARDSYDSSLYVGDYFDY DFAVYYCQQRSNWPLTFGQ WGQGTLVTVSSASTKGPSVFPLA GTKVEIKRTVAAPSVFIFPPS PCSRSTSESTAALGCLVKDYFPEP DEQLKSGTASVVCLLNNFYP VTVSWNSGALTSGVHTFPAVLQS REAKVQWKVDNALQSGNS SGLYSLSSVVTVPSSSLGTKTYTC QESVTEQDSKDSTYSLSSTLT NVDHKPSNTKVDKRVESKYGPPC LSKADYEKHKVYACEVTHQ PPCPAPEAAGGPSVFLFPPKPKDT GLSSPVTKSFNRGEC LMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK EVQLLESGGGLVQPGGSLRLSCA 108 QAVVTQEPSLTVSPGGTVTL 109 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYADSVK QKPGQAPRGLIGGTNKRAPG GRFTISRDNSKNTLYLQMNSLRA TPARFSGSLLGGKAATLTLSG EDTAVYYCVKHGNFGNSYVSWF AQPEDEAEYYCALWYSNLW AYWGQGTLVTVSSASTKGPSVFP VFGGGTKLTVLGQPKAAPS LAPCSRSTSESTAALGCLVKDYFP VTLFPPSSEELQANKATLVC EPVTVSWNSGALTSGVHTFPAVL LISDFYPGAVTVAWKADSSP QSSGLYSLSSVVTVPSSSLGTKTY VKAGVETTTPSKQSNNKYA TCNVDHKPSNTKVDKRVESKYGP ASSYLSLTPEQWKSHRSYSC PCPPCPAPEAAGGPSVFLFPPKPK QVTHEGSTVEKTVAPTECS DTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B10 EVQLLESGGGLVQPGGSLRLSCA  86 DIQMTQSPSSLSASVGDRVTI  87 (PSMB128 ASGFTFDSDWMSWVRQAPGKGL TCRASQSISSYLNWYQQKPG X EWVSAISGNGGSTEYADSVKGRF KAPKLLIYAASSLQSGVPSRF CD3B217) TISRDNSKNTLYLQMNSLRAEDT SGSGSGTDFTLTISSLQPEDF AVYYCARDPYYYYDGDSYYGM ATYYCQQSYSTPLTFGQGTK DVWGQGTLVTVSSASTKGPSVFP VEIKRTVAAPSVFIFPPSDEQ LAPCSRSTSESTAALGCLVKDYFP LKSGTASVVCLLNNFYPREA EPVTVSWNSGALTSGVHTFPAVL KVQWKVDNALQSGNSQESV QSSGLYSLSSVVTVPSSSLGTKTY TEQDSKDSTYSLSSTLTLSKA TCNVDHKPSNTKVDKRVESKYGP DYEKHKVYACEVTHQGLSS PCPPCPAPEAAGGPSVFLFPPKPK PVTKSFNRGEC DTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK EVQLLESGGGLVQPGGSLRLSCA 108 QAVVTQEPSLTVSPGGTVTL 109 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYADSVK QKPGQAPRGLIGGTNKRAPG GRFTISRDNSKNTLYLQMNSLRA TPARFSGSLLGGKAATLTLSG EDTAVYYCVKHGNFGNSYVSWF AQPEDEAEYYCALWYSNLW AYWGQGTLVTVSSASTKGPSVFP VFGGGTKLTVLGQPKAAPS LAPCSRSTSESTAALGCLVKDYFP VTLFPPSSEELQANKATLVC EPVTVSWNSGALTSGVHTFPAVL LISDFYPGAVTVAWKADSSP QSSGLYSLSSVVTVPSSSLGTKTY VKAGVETTTPSKQSNNKYA TCNVDHKPSNTKVDKRVESKYGP ASSYLSLTPEQWKSHRSYSC PCPPCPAPEAAGGPSVFLFPPKPK QVTHEGSTVEKTVAPTECS DTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B11 QVQLVQSGAEVKKPGSSVKVSCK  82 DIQMIQSPSSLSASVGDRVTI  83 (PSMB129 ASGGTFSSYAISWVRQAPGQGLE TCRASQSIDRWLNWYQQKP X WMGWISPYNGNANYAQKFQGRV GKAPKLLIYAASSLQSGVPS CD3B217) TITADESTSTAYMELSSLRSEDTA RFSGSGSGTDFTLTISSLQPE VYYCARVNSAALVWERLDYWG DFATYYCQQSPRYPLTFGQG QGTLVTVSSASTKGPSVFPLAPCS TKVEIKRTVAAPSVFIFPPSD RSTSESTAALGCLVKDYFPEPVTV EQLKSGTASVVCLLNNFYPR SWNSGALTSGVHTFAVLQSSGL EAKVQWKVDNALQSGNSQE YSLSSVVTVPSSSLGTKTYTCNVD SVTEQDSKDSTYSLSSTLTLS HKPSNTKVDKRVESKYGPPCPPC KADYEKHKVYACEVTHQGL PAPEAAGGPSVFLFPPKPKDTLMI SSPVTKSFNRGEC SRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPRELQFNS TYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQREMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALEINHY TQKSLSLSLGK EVQLLESGGGLVQPGGSLRLSCA 108 QAVVTQEPSLTVSPGGTVTL 109 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYADSVK QKPGQAPRGLIGGTNKRAPG GRFTISRDNSKNTLYLQMNSLRA TPARFSGSLLGGKAATLTLSG EDTAVYYCVKHGNFGNSYVSWF AQPEDEAEYYCALWYSNLW AYWGQGTLVTVSSASTKGPSVFP VFGGGTKLTVLGQPKAAPS LAPCSRSTSESTAALGCLVKDYFP VTLFPPSSEELQANKATLVC EPVTVSWNSGALTSGVHTFPAVL LISDFYPGAVTVAWKADSSP QSSGLYSLSSVVTVPSSSLGTKTY VKAGVETTTPSKQSNNKYA TCNVDHKPSNTKVDKRVESKYGP ASSYLSLTPEQWKSHRSYSC PCPPCPAPEAAGGPSVFLFPPKPK QVTHEGSTVEKTVAPTECS DTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B12 QVQLVQSGAEVKKPGSSVKVSCK  84 EIVLTQSPGTLSLSPGERATL  85 (PSMB130 ASGGTFKSYDISWVRQAPGQGLE SCRASQSVSSSYLAWYQQKP X WMGGIIPIEGTANYAQKFQGRVTI GQAPRLLIYGASSRATGIPDR CD3B217) TADESTSTAYMELSSLRSEDTAV FSGSGSGTDFTLTISRLEPED YYCARDYPAGYGFDYWGQGTLV FAVYYCQQYGSSPLTFGQGT TVSSASTKGPSVFPLAPCSRSTSES KVEIKRTVAAPSFIFPPSDE TAALGCLVKDYFPEPVTVSWNSG QLKSGTASVVCLLNNGYPRE ALTSGVHTFPAVLQSSGLYSLSSV AKVQWKVDNALQSGNSQES VTVPSSSLGTKTYTCNVDHKPSN VTEQDSKDSTYSLSSTLTLSK TKVDKRVESKYGPPCPPCPAPEA ADYRKHKVYACEVTHQGLS AGGPSVFLFPPKPKDTLMISRTPE SPVTKSFNRGEC VTCVVVDVSQEDPEVQFNWVVD GVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMEEALHNHYTQKSL SLSLGK EVQLLESGGGLVQPGGSLRLSCA 108 QAVVTQEPSLTVSPGGTVTL 109 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYADSVK QKPGQAPRGLIGGTNKRAPG GRFTISRDNSKNTLYLQMNSLRA TPARFSGSLLGGKAATLTLSG EDTAVYYCVKHGNFGNSYVSWF AQPEDEAEYYCALWYSNLW AYWGQGTLVTVSSASTKGPSVFP VFGGGTKLTVLGQPKAAPS LAPCSRSTSESTAALGCLVKDYFP VTLFPPSSEELQANKATLVC EPVTVSWNSGALTSGVHTFPAVL LISDFYPGAVTVAWKADSSP QSSGLYSLSSVVTVPSSSLGTKTY VKAGVETTTPSKQSNNKYA TCNVDHKPSNTKVDKRVESKYGP ASSYLSLTPEQWKSHRSYSC PCPPCPAPEAAGGPSVFLFPPKPK QVTHEGSTVEKTVAPTECS DTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B11 QVQLVQSGAEVKKPGSSVKVSCK  82 DIQMIQSPSSLSASVGDRVTI  83 (PSMB129 ASGGTFSSYAISWVRQAPGQGLE TCRASQSIDRWLNWYQQKP X WMGWISPYNGNANYAQKFQGRV GKAPKLLIYAASSLQSGVPS CD3B217) TITADESTSTAYMELSSLRSEDTA RFSGSGSGTDFTLTISSLQPE VYYCARVNSAALVWERLDYWG DFATYYCQQSPRYPLTFGQG QGTLVTVSSASTKGPSVFPLAPCS TKVEIKRTVAAPSVFIFPPSD RSTSESTAALGCLVKDYFPEPVTV EQLKSGTASVVCLLNNFYPR SWNSGALTSGVHTFAVLQSSGL EAKVQWKVDNALQSGNSQE YSLSSVVTVPSSSLGTKTYTCNVD SVTEQDSKDSTYSLSSTLTLS HKPSNTKVDKRVESKYGPPCPPC KADYEKHKVYACEVTHQGL PAPEAAGGPSVFLFPPKPKDTLMI SSPVTKSFNRGEC SRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPRELQFNS TYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQREMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALEINHY TQKSLSLSLGK EVQLVESGGGLVQPGGSLRLSCA 110 QTVVTQEPSLTVSPGGTVTL 111 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYAASVK QKPGQAPRGLIGGTNKRAPG GRFTISRDDSKNSLYLQMNSLKTE TPARFSSGSLLGGKAALTLSG DTAVYYCARHGNFGNSYVSWFA VQPEDEAEYYCALWYSNLW YWGQGTLVTVSSASTKGPSVFPL VFGGGTKLTVLGQPKAAPS APCSRSTSESTAALGCLVKDYFPE VTLFPPSSEELQANKATLVC PVTVSWNSGALTSGVHTFPAVLQ LISDFYPGAVTVAWKADSSP SSGLYSLSSVVTVPSSSLGTKTYT VKAGVETTTPSKQSNNKYA CNVDHKPSNTKVDKRVESKYGPP ASSYLSLTPEQWKSHRSYSC CPPCPAPEAAGGPSVFLFPPKPKD QVTHEGSTVEKTVAPTECS TLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B30 QVQLVQSGAEVKKPGSSVKVSCK  84 EIVLTQSPGTLSLSPGERATL  85 (PSMB130 ASGGTFKSYDISWVRQAPGQGLE SCRASQSVSSSYLAWYQQKP X WMGGIIPIEGTANYAQKFQGRVTI GQAPRLLIYGASSRATGIPDR CD3B219) TADESTSTAYMELSSLRSEDTAV FSGSGSGTDFTLTISRLEPED YYCARDYPAGYGFDYWGQGTLV FAVYYCQQYGSSPLTFGQGT TVSSASTKGPSVFPLAPCSRSTSES KVEIKRTVAAPSVFIFPPSDE TAALGCLVKDYFPEPVTVSWNSG QLKSGTASVVCLLNNFYPRE ALTSGVHTFPAVLQSSGLYSLSSV AKVQWKVDNALQSGNSQES VTVPSSSLGTKTYTCNVDHKPSN VTEQDSKDSTYSLSSTLTLSK TKVDKRVESKYGPPCPPCPAPEA ADYEKHKVYACEVTHQGLS AGGPSVPLFPPKPKDTLMISRTPE SPVTKSFNRGEC VTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSL SLSLGK EVQLVESGGGLVQPGGSLRLSCA 110 QTVVTQEPSLTVSPGGTVTL 111 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYAASVK QKPGQAPRGLIGGTNKRAPG GRFTISRDDSKNSLYLQMNSLKTE TPARFSSGSLLGGKAALTLSG DTAVYYCARHGNFGNSYVSWFA VQPEDEAEYYCALWYSNLW YWGQGTLVTVSSASTKGPSVFPL VFGGGTKLTVLGQPKAAPS APCSRSTSESTAALGCLVKDYFPE VTLFPPSSEELQANKATLVC PVTVSWNSGALTSGVHTFPAVLQ LISDFYPGAVTVAWKADSSP SSGLYSLSSVVTVPSSSLGTKTYT VKAGVETTTPSKQSNNKYA CNVDHKPSNTKVDKRVESKYGPP ASSYLSLTPEQWKSHRSYSC CPPCPAPEAAGGPSVFLFPPKPKD QVTHEGSTVEKTVAPTECS TLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B28 EVQLLESGGGLVQPGGSLRLSCA  86 DIQMTQSPSSLSASVGDRVTI  87 (PSMB128 ASGFTFDSDWMSWVRQAPGKGL TCRASQSISSYLNWYQQKPG X EWVSAISGNGGSTEYADSVKGRF KAPKLLIYAASSLQSGVPSRF CD3B219) TISRDNSKNTLYLQMNSLRAEDT SGSGSGTDFTLTISSLQPEDF AVYYCARDPYYYYDGDSYYGM ATYYCQQSYSTPLTFGQGTK DVWGQGTLVTVSSASTKGPSVFP VEIKRTVAAPSVFIEPPSDEQ LAPCSRSTSESTAALGCLVKDYFP LKSGTASVVCLLNNFYPREA EPVTVSWNSGALTSGVHTFPAVL KVQWKVDNALQSGNSQESV QSSGLYSLSSVVTVPSSSLGTKTY TEQDSKDSTYSLSSTLTLSKA TCNVDHKPSNTKVDKRVESKYGP DYEKHKVYACEVTHQGLSS PCPPCPAPEAAGGPSVFLFPPKPK PVTKSFNRGEC DTLMISRTREVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK EVQLVESGGGLVQPGGSLRLSCA 110 QTVVTQEPSLTVSPGGTVTL 111 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYAASVK QKPGQAPRGLIGGTNKRAPG GRFTISRDDSKNSLYLQMNSLKTE TPARFSSGSLLGGKAALTLSG DTAVYYCARHGNFGNSYVSWFA VQPEDEAEYYCALWYSNLW YWGQGTLVTVSSASTKGPSVFPL VFGGGTKLTVLGQPKAAPS APCSRSTSESTAALGCLVKDYFPE VTLFPPSSEELQANKATLVC PVTVSWNSGALTSGVHTFPAVLQ LISDFYPGAVTVAWKADSSP SSGLYSLSSVVTVPSSSLGTKTYT VKAGVETTTPSKQSNNKYA CNVDHKPSNTKVDKRVESKYGPP ASSYLSLTPEQWKSHRSYSC CPPCPAPEAAGGPSVFLFPPKPKD QVTHEGSTVEKTVAPTECS TLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B27 EVQLLESGGGLVQPGGSLRLSCA  88 EIVLTQSPATLSLSPGERATL  89 (PSMB127 ASGFTFKSDAMEWVRQAPGKGL SCRASQSVSSYLAWYQQKP X EWVSEISGSGGYTNYADSVKGRF GQAPRLLIYDASNRATGIPA CD3B219) TISRDNSKNTLYQMNSLRAEDT RFSGSGSGTDFTLTISSLEPE AVYYCARDSYDSSLYVGDYFDY DFAVYYCQQRSNWPLTFGQ WGQGTLVTVSSASTKGPSVFPLA GTKVEIKRTVAAPSVFIFPPS PCSRSTSESTAALGCLVKDYFPEP DEQLKSGTASVVCLLNNFYP VTVSWNSGALTSGVHTFPAVLQS REAKVQWKVDNALQSGNS SGLYSLSSVVTVPSSSLGTKTYTC QESVTEQDSKDSTYSLSSTLT NVDHKPSNTKVDKRVESKYGPPC LSKADYEKHKVYACEVTHQ PPCPAPEAAGGPSVFLFPPKPKDT GLSSPVTKSFNRGEC LMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK EVQLVESGGGLVQPGGSLRLSCA 110 QTVVTQEPSLTVSPGGTVTL 111 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYAASVK QKPGQAPRGLIGGTNKRAPG GRFTISRDDSKNSLYLQMNSLKTE TPARFSSGSLLGGKAALTLSG DTAVYYCARHGNFGNSYVSWFA VQPEDEAEYYCALWYSNLW YWGQGTLVTVSSASTKGPSVFPL VFGGGTKLTVLGQPKAAPS APCSRSTSESTAALGCLVKDYFPE VTLFPPSSEELQANKATLVC PVTVSWNSGALTSGVHTFPAVLQ LISDFYPGAVTVAWKADSSP SSGLYSLSSVVTVPSSSLGTKTYT VKAGVETTTPSKQSNNKYA CNVDHKPSNTKVDKRVESKYGPP ASSYLSLTPEQWKSHRSYSC CPPCPAPEAAGGPSVFLFPPKPKD QVTHEGSTVEKTVAPTECS TLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B26 QVQLVQSGAEVKKPGSSVKVSCK  90 DIQMTQSPSSLSASVGDRVTI  91 (PSMB126 ASGGTFDDYAISWVRQAPGQGLE TCRASQSISSYLNWYQQKPG X WMGRIDPIEGTANYAQKFQGRVT KAPKILIYAASSLQSGVPSRF CD3B219) ITADESTSTAYMELSSLRSEDTAV SGSGSGTDFTLTISSLQPEDF YYCARDRYYYDGVYWYSDYFD ATYYCQQSYSTPLTFGQGTK YWGQGTLVTVSSASTKGPSVFPL VEIKRTVAAPSVFIFPPSDEQ APCSRSTSESTAALGCLVKDYFPE LKSGTASVVCLLNNFYPREA PVTVSWNSGALTSGVHTFPAVLQ KVQWKVDNALQSGNSQESV SSGLYSLSSVVTVPSSSLGTKTYT TEQDSKDSTYSLSSTLTLSKA CNVDHKPSNTKVDKRVESKYGPP DYEKHKVYACEVTHQGLSS CPPCPAPEAAGGPSVFLEPPKPKD PVTKSFNRGEC TLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK EVQLVESGGGLVQPGGSLRLSCA 110 QTVVTQEPSLTVSPGGTVTL 111 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYAASVK QKPGQAPRGLIGGTNKRAPG GRFTISRDDSKNSLYLQMNSLKTE TPARFSSGSLLGGKAALTLSG DTAVYYCARHGNFGNSYVSWFA VQPEDEAEYYCALWYSNLW YWGQGTLVTVSSASTKGPSVFPL VFGGGTKLTVLGQPKAAPS APCSRSTSESTAALGCLVKDYFPE VTLFPPSSEELQANKATLVC PVTVSWNSGALTSGVHTFPAVLQ LISDFYPGAVTVAWKADSSP SSGLYSLSSVVTVPSSSLGTKTYT VKAGVETTTPSKQSNNKYA CNVDHKPSNTKVDKRVESKYGPP ASSYLSLTPEQWKSHRSYSC CPPCPAPEAAGGPSVFLFPPKPKD QVTHEGSTVEKTVAPTECS TLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B25 EVQLVQSGAEVKKPGESLKISCK 125 DIQMTQSPSSLSASVGDRVTI  91 (PSMB87 GSGYSFTSYWISWVRQMPGKGLE TCRASQSISSYLNWYQQKPG X WMGITYPGDSYTRYSPSFQGQVTI KAPKILIYAASSLQSGVPSRF CD3B219) SADKSISTAYLQWSSLKASDTAM SGSGSGTDFTLTISSLQPEDF YYCARDYEWELFDSRLDYWGQG ATYYCQQSYSTPLTFGQGTK TLVTVSSASTKGPSVFPLAPCSRS VEIKRTVAAPSVFIFPPSDEQ TSESTAALGCLVKDYFPEPVTVS LKSGTASVVCLLNNFYPREA WNSGALTSGVHTFPAVLQSSGLY KVQWKVDNALQSGNSQESV SLSSVVTVPSSSLGTKTYTCNVDH TEQDSKDSTYSLSSTLTLSKA KPSNTKVDKRVESKYGPPCPPCP DYEKHKVYACEVTHQGLSS APEAAGGPSVFLFPPKPKDTLMIS PVTKSFNRGEC RTPEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK EVQLVESGGGLVQPGGSLRLSCA 110 QTVVTQEPSLTVSPGGTVTL 111 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYAASVK QKPGQAPRGLIGGTNKRAPG GRFTISRDDSKNSLYLQMNSLKTE TPARFSSGSLLGGKAALTLSG DTAVYYCARHGNFGNSYVSWFA VQPEDEAEYYCALWYSNLW YWGQGTLVTVSSASTKGPSVFPL VFGGGTKLTVLGQPKAAPS APCSRSTSESTAALGCLVKDYFPE VTLFPPSSEELQANKATLVC PVTVSWNSGALTSGVHTFPAVLQ LISDFYPGAVTVAWKADSSP SSGLYSLSSVVTVPSSSLGTKTYT VKAGVETTTPSKQSNNKYA CNVDHKPSNTKVDKRVESKYGPP ASSYLSLTPEQWKSHRSYSC CPPCPAPEAAGGPSVFLFPPKPKD QVTHEGSTVEKTVAPTECS TLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B24 QVQLVQSGAEVKKPGSSVKVSCK  92 DIQMTQSPSSLSASVGDRVTI  93 (PSMB124 ASGGTFSSYAISWVRQAPGQGLE TCRASQSISGWLNWYQQKP X WMGWISPYNGNANYAQKFQGRV GKAPKLLIYAASSLQSGVPS CD3B219) TITADESTSTAYMELSSLRSEDTA RFSGSGSGTDFTLTISSLQPE VYYCARDSDRSYNLDYWGQGTL DFATYYCQQSYSTPLTFGQG VTVSSASTKGPSVFPLAPCSRSTSE TKVEIKRTVAAPSVFIFPPSD STAALGCLVKDYFPEPVTVSWNS EQLKSGTASVVCLLNNEYTR GALTSGVHTFPAVLQSSGLYSLSS EAKVQWKVDNALQSGNSQE VVTVPSSSLGTKTYTCNVDHKPS SVTEQDSKDSTYSLSSTLTLS NTKVDKRVESKYGPPCPPCPAPE KADYEKHKVYACEVTHQGL AAGGPSVFLFPPKPKDTLMISRTP SSPVTKSFNRGEC EVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVS NKGLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQK SLSLSLGK EVQLVESGGGLVQPGGSLRLSCA 110 QTVVTQEPSLTVSPGGTVTL 111 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYAASVK QKPGQAPRGLIGGTNKRAPG GRFTISRDDSKNSLYLQMNSLKTE TPARFSSGSLLGGKAALTLSG DTAVYYCARHGNFGNSYVSWFA VQPEDEAEYYCALWYSNLW YWGQGTLVTVSSASTKGPSVFPL VFGGGTKLTVLGQPKAAPS APCSRSTSESTAALGCLVKDYFPE VTLFPPSSEELQANKATLVC PVTVSWNSGALTSGVHTFPAVLQ LISDFYPGAVTVAWKADSSP SSGLYSLSSVVTVPSSSLGTKTYT VKAGVETTTPSKQSNNKYA CNVDHKPSNTKVDKRVESKYGPP ASSYLSLTPEQWKSHRSYSC CPPCPAPEAAGGPSVFLFPPKPKD QVTHEGSTVEKTVAPTECS TLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B23 EVQLVQSGAEVKKPGESLKISCK  94 EIVLTQSPATLSLSPGERATL  95 (PSMB123 GSGYSFTSYWIGWVRQMPGKGL SCRASQSVASDLAWYQQKP X EWMGIIYPGDSDTRYSPSFQGQVT GQAPRLLIYFASNRATGIPAR (D3B219) ISADKSISTAYLQWSSLKASDTAM FSGSGSGTDFTLTISSLEPEDF YYCARGLPIWYLDYWGQGTLVT AVVYCQQSITWPFTFGQGTK VSSASTKGPSVFPLAPCSRSTSEST VEIKRTVAAPSVFIFPPSDEQ AALGCLVKDYTPEPVTVSWNSGA LKSGTASVVCLLNNFYPREA LTSGVHTFPAVLQSSGLYSLSSVV KVQWKVDNALQSGNSQESV TVPSSSLGTKTYTCNVDHKPSNT TEQDSKDSTYSLSSTLTLSKA KVDKRVESKYGPPCPPCPAPEAA DYEKHKVYACEVTHQGLSS GGPSVFLFPPKPKDTLMISRTPEV PVTKSFNRGEC TCVVVDVSQEDREVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVS VLTVLHWDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSL SLGK EVQLVESGGGLVQPGGSLRLSCA 110 QTVVTQEPSLTVSPGGTVTL 111 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYAASVK QKPGQAPRGLIGGTNKRAPG GRFTISRDDSKNSLYLQMNSLKTE TPARFSSGSLLGGKAALTLSG DTAVYYCARHGNFGNSYVSWFA VQPEDEAEYYCALWYSNLW YWGQGTLVTVSSASTKGPSVFPL VFGGGTKLTVLGQPKAAPS APCSRSTSESTAALGCLVKDYFPE VTLFPPSSEELQANKATLVC PVTVSWNSGALTSGVHTFPAVLQ LISDFYPGAVTVAWKADSSP SSGLYSLSSVVTVPSSSLGTKTYT VKAGVETTTPSKQSNNKYA CNVDHKPSNTKVDKRVESKYGPP ASSYLSLTPEQWKSHRSYSC CPPCPAPEAAGGPSVFLFPPKPKD QVTHEGSTVEKTVAPTECS TLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B22 QVQLVQSGAEVKKPGSSVKVSCK  96 DIQMTQSPSSLSASVGDRVTI  83 (PSMB123 ASGGTFSSYAISWVRQAPGQGLE TCRASQSIDRWLNWYQQKP X WMGWIIPYNGNANYAQKFQGRV GKAPKLLIYAASSLQSGVPS CD3B219) TITADESTSTAYMELSSLRSEDTA RFSGSGSGTDFTLTISSLQPE VYYCARVNSAALVWERLDYWG DFATYYCQQSPRYPLTFGQG QGTLVTVSSASTKGPSVFPLAPCS TKVEIKRTVAAPSVFIFPPSD RSTSESTAALGCLVKDYFPEPVTV EQLKSGTASVVCLLNNFYPR SWNSGALTSGVHTFPAVLQSSGL EAKVQWKVDNALQSGNSQE YSLSSVVTVPSSSLGTKTYTCNVD SVTEQDSKDSTYSLSSTLTLS HKPSNTKVDKRVESKYGPPCPPC KADYEKHKVYACEVTHQGL PAPEAAGGPSVFLFPPKPKDTLMI SSPVTKSFNRGEC SRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNS TYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK EVQLVESGGGLVQPGGSLRLSCA 110 QTVVTQEPSLTVSPGGTVTL 111 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYAASVK QKPGQAPRGLIGGTNKRAPG GRFTISRDDSKNSLYLQMNSLKTE TPARFSSGSLLGGKAALTLSG DTAVYYCARHGNFGNSYVSWFA VQPEDEAEYYCALWYSNLW YWGQGTLVTVSSASTKGPSVFPL VFGGGTKLTVLGQPKAAPS APCSRSTSESTAALGCLVKDYFPE VTLFPPSSEELQANKATLVC PVTVSWNSGALTSGVHTFPAVLQ LISDFYPGAVTVAWKADSSP SSGLYSLSSVVTVPSSSLGTKTYT VKAGVETTTPSKQSNNKYA CNVDHKPSNTKVDKRVESKYGPP ASSYLSLTPEQWKSHRSYSC CPPCPAPEAAGGPSVFLFPPKPKD QVTHEGSTVEKTVAPTECS TLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B21 QVQLVQSGAEVKKPGSSVKVSCK  97 EIVLTQSPATLSLSPGERATL  98 (PSMB121 ASGGTFSSYAISWVRQAPGQGLE SCRASQSVSKWLAWYQQKP X WMGGIIPIFGTANYAQKFQGRVTI GQAPRLLIYDASNRATGIPA CD3B219) TADESTSTAYMELSSLRSEDTAV RFSGSGSGTDFTLTISSLEPE YYCARASRVWHASYGYLDYWG DFAVYYCQQRFTAPWTFGQ QGTLVTVSSASTKGPSVFPLAPCS GTKVEIKRTVAAPSVFIFPPS RSTSESTAALGCLVKDYFPEPVTV DEQLKSGTASVVCLLNNFYP SWNSGALTSGVHTFPAVLQSSGL REAKVQWKVDNALQSGNS YSLSSVVTVPSSSLGTKTYTCNVD QESVTEQDSKDSTYSLSSTLT HKPSNTKVDKRVESKYGPPCPPC LSKADYEKHKVYACEVTHQ PAPEAAGGPSVFLYPPKPKDTLMI GLSSPVTKSFNRGEC SRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNS TYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK EVQLVESGGGLVQPGGSLRLSCA 110 QTVVTQEPSLTVSPGGTVTL 111 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYAASVK QKPGQAPRGLIGGTNKRAPG GRFTISRDDSKNSLYLQMNSLKTE TPARFSSGSLLGGKAALTLSG DTAVYYCARHGNFGNSYVSWFA VQPEDEAEYYCALWYSNLW YWGQGTLVTVSSASTKGPSVFPL VFGGGTKLTVLGQPKAAPS APCSRSTSESTAALGCLVKDYFPE VTLFPPSSEELQANKATLVC PVTVSWNSGALTSGVHTFPAVLQ LISDFYPGAVTVAWKADSSP SSGLYSLSSVVTVPSSSLGTKTYT VKAGVETTTPSKQSNNKYA CNVDHKPSNTKVDKRVESKYGPP ASSYLSLTPEQWKSHRSYSC CPPCPAPEAAGGPSVFLFPPKPKD QVTHEGSTVEKTVAPTECS TLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B20 EVQLVQSGAEVKKPGESLKISCK  99 DIVMTQSPDSLAVSLGERATI 100 (PSMB120 GSGYSFTSYWIGWVRQMPGKGL NCKSSQSVLYSSNNKNYLA X EWMGIIYPGDSDTRYSPSFQGQVT WYQQKPGQPPKLLIYWAST CD3B219) ISADKSISTAYLQWSSLKASDTAM RESGVPDRFSGSGSGTDFTL YYCARGWAYDRGLDYWGQGTL TISSLQAEDVAVYYCQQYYS VTVSSASTKGPSVFPLAPCSRSTSE TPLTFGQGTKVEIKRTVAAP STAALGCLVKDYFPEPVTVSWNS SVFIFPPSDEQLKSGTASVVC GALTSGVHTFPAVLQSSGLYSLSS LLNNFYPREAKVQWKVDNA VVTVPSSSLGTKTYTCNVDHKPS LQSGNSQESVTEQDSKDSTY NTKVDKRVESKYGPPCPPCPAPE SLSSTLTLSKADYEKHKVYA AAGGPSVFLFPPKPKDTLMISRTP CEVTHQGLSSPVTKSPNRGE EVTCVVVDVSQEDPEVQFNWYV C DGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVS NKGLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQK SLSLSLGK EVQLVESGGGLVQPGGSLRLSCA 110 QTVVTQEPSLTVSPGGTVTL 111 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYAASVK QKPGQAPRGLIGGTNKRAPG GRFTISRDDSKNSLYLQMNSLKTE TPARFSSGSLLGGKAALTLSG DTAVYYCARHGNFGNSYVSWFA VQPEDEAEYYCALWYSNLW YWGQGTLVTVSSASTKGPSVFPL VFGGGTKLTVLGQPKAAPS APCSRSTSESTAALGCLVKDYFPE VTLFPPSSEELQANKATLVC PVTVSWNSGALTSGVHTFPAVLQ LISDFYPGAVTVAWKADSSP SSGLYSLSSVVTVPSSSLGTKTYT VKAGVETTTPSKQSNNKYA CNVDHKPSNTKVDKRVESKYGPP ASSYLSLTPEQWKSHRSYSC CPPCPAPEAAGGPSVFLFPPKPKD QVTHEGSTVEKTVAPTECS TLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK PS3B19 EVQLVQSGAEVKKPGESLKISCK 101 DIVMTQSPDSLAVSLGERATI 100 (PSMB119 GSGYSETSYWIGWVRQMPGKGL NCKSSQSVLYSSNNKNYLA X EWMGIIYPGDSDTRYSPSFQGQVT WYQQKPGQPPKLLIYWAST CD3B219) ISADKSISTAYLQWSSLKASDTAM RESGVPDRFSGSGSGTDFTL YYCARAYHYSKGLDYWGQGTLV TISSLQAEDVAVYYCQQYYS TVSSASTKGPSVFPLAPCSRSTSES TPLTFGQGTKVEIKRTVAAP TAALGCLVKDYFPEPVTVSWNSG SVFIFPPSDEQLKSGTASVVC ALTSGVHTFPAVLQSSGLYSLSSV LLNNFYPREAKVQWKVDNA VTVPSSSLGTKTYTCNVDHKPSN LQSGNSQESVTEQDSKDSTY TKVDKRVESKYGPPCPPCPAPEA SLSSTLTLSKADYEKHKVYA AGGPSVFLFPPKPKDTLMISRTPE CEVTHQGLSSPVTKSFNRGE VTCVVVDVSQEDPEVQFNWYVD C GVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSL SLSLGK EVQLVESGGGLVQPGGSLRLSCA 110 QTVVTQEPSLTVSPGGTVTL 111 ASGFTFNTYAMNWVRQAPGKGL TCRSSTGAVTTSNYANWVQ EWVARIRSKYNNYATYYAASVK QKPGQAPRGLIGGTNKRAPG GRFTISRDDSKNSLYLQMNSLKTE TPARFSSGSLLGGKAALTLSG DTAVYYCARHGNFGNSYVSWFA VQPEDEAEYYCALWYSNLW YWGQGTLVTVSSASTKGPSVFPL VFGGGTKLTVLGQPKAAPS APCSRSTSESTAALGCLVKDYFPE VTLFPPSSEELQANKATLVC PVTVSWNSGALTSGVHTFPAVLQ LISDFYPGAVTVAWKADSSP SSGLYSLSSVVTVPSSSLGTKTYT VKAGVETTTPSKQSNNKYA CNVDHKPSNTKVDKRVESKYGPP ASSYLSLTPEQWKSHRSYSC CPPCPAPEAAGGPSVFLFPPKPKD QVTHEGSTVEKTVAPTECS TLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK

TABLE 12 VH/VL Sequences of PSMA × CD3 bispecific antibodies SEQ ID NO: (VH; VL IgG4PAA F405L, Bispecific SEQ ID NO: R409K) antibody ARM 1 VH VL ARM 2 VH VL PS3B2 PSMB120 77 78 CD3B217 102 103 (FAB PSMB25) PS3B3 PSMB121 75 76 CD3B217 102 103 (FAB PSMB49) PS3B4 PSMB122 74 61 CD3B217 102 103 (FAB PSMB51) PS3B5 PSMB123 75 76 CD3B217 102 103 (FAB PSMB55) PS3B7 PSMB87 160 64 CD3B217 102 103 (FAB PSMB58) PS3B8 PSMB126 68 69 CD3B217 102 103 (FAB PSMB80) PS3B9 PSMB127 66 67 CD3B217 102 103 (FAB PSMB83) PS3B10 PSMB128 64 65 CD3B217 102 103 (FAB PSMB84) PS3B11 PSMB129 60 61 CD3B217 102 103 (FAB PSMB109) PS3B12 PSMB130 62 63 CD3B217 102 103 (FAB PSMB86) PS3B19 PSMB119 79 78 CD3B219 104 105 (FAB PSMB18) PS3B20 PSMB120 77 178 CD3B219 104 105 (FAB PSMB25) PS3B21 PSMB121 75 76 CD3B219 104 105 (FAB PSMB49) PS3B22 PSMB122 74 61 CD3B219 104 105 (FAB PSMB51) PS3B23 PSMB123 75 76 CD3B219 104 105 (FAB PSMB55) PS3B24 PSMB124 70 71 CD3B219 104 105 (FAB PSMB56) PS3B25 PSMB87 160 65 CD3B219 104 105 (FAB PSMB58) PS3B26 PSMB126 68 69 CD3B219 104 105 (FAB PSMB80) PS3B27 PSMB127 66 67 CD3B219 104 105 (FAB PSMB83) PS3B28 PSMB128 64 65 CD3B219 104 105 (FAB PSMB84) PS3B29 PSMB129 60 61 CD3B219 104 105 (FAB PSMB109) PS3B30 PSMB130 62 63 CD3B219 104 105 (FAB PSMB86)

Example 7. Characterization by Cell-Binding

PSMA×CD3 bispecific antibodies were tested for binding to PSMA positive cell lines LNCAP, human PSMA-HEK, Chimpanzee-PSMA-HEK and Cynomolgous monkey PSMA-HEK. To assess the binding capabilities of the PSMA bispecific antibodies, the cell-binding assay was utilized (described previously). Briefly, PSMA expressing tumor cells are bound by bispecific antibodies at known concentrations and the bound antibody is detected by an anti-human kappa light chain PE conjugated detection reagent (Invitrogen). The Mean Fluorescents Intensity (MFI) is the measure of bound bispecific antibody. The MFI is converted to a relative EC₅₀. EC₅₀ is a commonly used dose-response curve, where the half maximal effective concentration or the EC₅₀ point is defined as the inflection point of the curve. EC₅₀ s were determined by measuring cell bound bispecific and known concentrations. High concentrations resulted in maximum target antigen binding i.e. full binding saturation. The dose response curves were then diluted down to that of background or no bispecific binding. The inflection point of this curve reflects the EC₅₀ point. The calculated EC₅₀ is determined by taking the ug/ml amount of bispecific antibody at the EC₅₀ point and converting it to a molarity value based on the MW of the bispecific antibody. Bispecific antibodies were normalized for protein concentration and then incubated with the same number of cells expressing either human or cyno PSMA. The MFI at each concentration was collected by flow cytometry and plotted as a function of concentration. Data was transformed via log 10 and then plotted. Nonlinear regression of binding curves was done to determine EC₅₀ s. These relative values were used for ranking PSMA binding to target cells. Table 12 contains the relative EC₅₀ binding values for whole cell binding studies using LNCaP, cyno and chimp PSMA-expressing cell lines.

FIG. 11 shows LNCAP binding of all bispecific antibodies prepared. The binding data suggests that 3 populations of binding are observed: 1) strong binding, 2) medium binding, and 3) weak/no binding. The PSMA X null arm bispecific antibodies maintain binding to LNCAP cells (FIG. 11E), but there is no binding observed for the null X CD3 arm bispecific antibodies (FIG. 11F). The medium affinity and high affinity CD3 arm bispecific antibodies with the same PSMA arm bind similarly. For the remaining assays, only bispecific antibodies that were positive LNCaP binders were used to assess binding activity to Chimp PSMA-HEK (FIG. 12), Cyno PSMA-HEK (FIG. 13), Human-PSMA-HEK (FIG. 14), or Parental HEK293 (data not shown). For each cell line, either the high-affinity or medium affinity CD3 binders were tested. The hPSMA-HEK binding data suggests that there may be subtle differences between LNCaP and this cell line; however, the same overall order of binding is evident. PS3B19, derived from LNCaP panning, appears to bind as strongly to hPSMA-HEK cells. The bispecific antibodies show a wide range of binding profiles on Chimp-PSMA HEK. Interestingly, hits that were panned on the Chimp-PSMA HEK cell line have a stronger binding profile, while those that were panned on LNCaP show weaker binding. There was no binding observed to the parental HEK cells in this experiment (data not shown).

Following recombination into bispecific antibodies, several clones consistently outperform others and bind cross-species. These bispecific antibodies are PS3B21, PS3B22, PS3B26, PS3B27, PS3B28 and PS3B30 which correspond to mAbs PSMB121, PSMB122, PSMB126, PSMB127, PSMB128 and PSMB130. Cell based binding EC₅₀'s and calculated EC₅₀'s are shown in Table 13.

TABLE 13 Cell Based Binding EC₅₀'s. LNCaP Cyno Chimp Calcu- Calcu- Calcu- lated lated lated EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ bsAb (ug/mL) (nM) (ug/mL) (nM) (ug/mL) (nM) PS3B19 too weak N/A 1.757 12.4 5.10 35.93 PS3B20 122.7 864.1 2.003 14.1 3.33 23.44 PS3B21 0.7368 5.2 0.8604 6.1 1.25 8.81 PS3B22 2.603 18.3 1.137 8 3.02 21.23 PS3B23 too weak N/A 3.744 26.4 0.88 6.18 PS3B24 too weak N/A 18.22 128.3 1.10 7.75 PS3B26 0.07147 0.5 0.3706 2.6 0.05 0.34 PS3B27 2.07 14.6 1.403 9.9 3.24 22.83 PS3B28 1.157 8.1 0.6879 4.8 1.01 7.08 PS3B29 7.766 54.7 2.539 17.9 4.11 28.93 PS3B30 1.203 8.5 5.37 37.8 3.29 23.19

All bispecific antibodies maintained the ability to bind PSMA positive cell lines. Several of the antibodies bound well to chimp and cyno PSMA-expressing cells, but only weakly to LNCaP cells. LNCAP binding EC₅₀'s ranged from 0.5 nM to 864 nM, whereas Cyno PSMA expressing HEK binding EC₅₀'s ranged from 0.9 to 128 nM and Chimp PSMA HEK binding ranged from 36-0.3 nM (Table 12). Based on cell binding EC₅₀'s several of the anti-PSMA bispecific antibodies met the criteria of 20 nM or tighter binding for human PSMA, and 50 nM or tighter binding for cyno PSMA.

Example 8. Affinity Characterization by Proteon and Biacore

To further evaluate the antibodies, the rates of chimp PSMA ECD association and dissociation were measured for the hits that were carried forward from Cell-binding assays.

The interactions of PSMA×CD3 bispecific mAbs with target (recombinant Chimp, PSMA) were studied by Surface Plasmon Resonance (SPR) using a ProteOn XPR36 system (BioRad). A biosensor surface was prepared by coupling anti-Human IgG Fc (Jackson ImmunoResearch Laboratory, cat #109-005-098) to the modified alginate polymer layer surface of a GLC chip (BioRad, cat #176-5011) using the manufacturer instructions for amine-coupling chemistry. Approximately 4400 RU (response units) of anti-Human IgG Fc antibodies were immobilized. The kinetic experiments were performed at 25° C. in running buffer (DPBS+0.03% P20+100 μg/ml BSA). To perform kinetic experiments, 100 RU of bispecific antibodies were captured followed by injections of analytes (recombinant Chimp PSMA ECD) at concentrations ranging from 3.7 nM to 300 nM (in a 3-fold serial dilution). The association phase was monitored for 3 minutes at 50 μL/min, then followed by 15 minutes of buffer flow (dissociation phase). The chip surface was regenerated with two 18 second pulses of 100 mM Phosphoric acid (H₃PO₄, Sigma, cat #7961) at 100 μL/min.

The collected data were processed using ProteOn Manager software. First, the data was corrected for background using inter-spots. Then, double reference subtraction of the data was performed by using the buffer injection for analyte injections. The kinetic analysis of the data was performed using a Langmuir 1:1 binding model. The result for each bispecific antibody was reported in the format of k_(a) (On-rate), k_(d) (Off-rate) and K_(D) (equilibrium dissociation constant). Results are shown in Tables 14-18.

Results:

TABLE 14 Summary of kinetics and affinity for PS3B25 and PS3B27 binding to recombinant human PSMA (3.7-300 nM). The parameters reported in this table were obtained from a 1:1 Langmuir binding model. Affinity, K_(D) = k_(d)/k_(a). Bispecific Ab k_(a) k_(d) K_(D) AKA Protein ID (1/Ms) 10⁵ (1/s) 10⁻⁰³ (nM) PSMB87 × PS3B25 1.88 ± 0.13 1.01 ± 0.05 5.38 ± 0.55 CD3B219 PSMB127 × PS3B27 2.87 ± 0.36 2.89 ± 0.70 10.3 ± 3.2  CD3B219 n = 3 independent experiments with 2 replicates. Results listed as average ± standard deviation.

TABLE 15 Summary of kinetics and affinity for PS3B25 and PS3B27 binding to recombinant chimp PSMA (3.7-300 nM). The parameters reported in this table were obtained from a 1:1 Langmuir binding model. Affinity, K_(D) = k_(d)/k_(a). Bispecific Ab k_(a) k_(d) K_(D) AKA Protein ID (1/Ms) 10⁵ (1/s) 10⁻⁰³ (nM) PSMB87 × PS3B25 2.81 ± 0.08 0.99 ± 0.04 3.54 ± 0 25 CD3B219 PSMB127 × PS3B27 2.08 ± 0.38 1.56 ± 0.37 7.48 ± 0.97 CD3B219 n = 3 independent experiments with 2 replicates. Results listed as average ± standard deviation.

TABLE 16 Summary of kinetics and affinity for PS3B25 and PS3B27 binding to recombinant cyno PSMA (3.7-300 nM). The parameters reported in this table were obtained from a 1:1 Langmuir binding model. Affinity, K_(D) = k_(d)/k_(a). Bispecific Ab k_(a) k_(d) K_(D) AKA Protein ID (1/Ms) 10⁵ (1/s) 10⁻⁰³ (nM) PSMB87 × PS3B25 0.98 ± 0.04 7.97 ± 0.34 81.1 ± 3.3  CD3B219 PSMB127 × PS3B27 1.59 ± 0.12 1.10 ± 0.04 7.00 ± 0.68 CD3B219 n = 3 independent experiments with 2 replicates. Results listed as average ± standard deviation.

TABLE 17 Comparing the Human, Chimp and Cyno binding affinity of PS3B25 and PS3B27. Affinity, K_(D) = k_(d)/k_(a). Human K_(D) Chimp K_(D) Cyno K_(D) Sample (nM) (nM) (nM) PS3B25 5.38 ± 0.55 3.54 ± 0.25 81.1 ± 3.3  PS3B27 10.3 ± 3.2  7.48 ± 0.97 7.00 ± 0.68

TABLE 18 Summary of kinetics and affinity for bispecific mAbs binding to recombinant chimp PSMA (3.7-300 nM). The parameters reported in this table were obtained from a 1:1 Langmuir binding model. Affinity, K_(D) = k_(d)/k_(a). Bispecific Ab Protein ID ka(1/Ms) kd(1/s) KD (nM) PS3B19 1.26E+04 2.98E−04 23.6 PS3B20 1.76E+04 3.78E−04 21.4 PS3B21 6.70E+04 4.73E−04 7.1 PS3B22 6.51E+04 1.24E−03 19 PS3B23 9.89E+05 1.76E−03 1.8 PS3B24 6.26E+03 2.52E−04 40.3 PS3B25 1.95E+05 1.06E−03 5.5 PS3B26 No binding PS3B27 1.70E+05 1.76E−03 10.4 PS3B28 1.341+05 2.33E−03 17.4 PS3B29 6.71E+04 6.80E−03 101 PS3B30 7.34E+04 1.65E−03 22.4 PS3B31 No binding PS3B32 No binding PS3B33 No binding PS3B34 No binding PSMA null No binding

For the most part, Proteon binding parallels the cell-binding. However, one of the bispecific antibodies showed no binding to recombinant chimp PSMA ECD although it bound to chimp PSMA expressed on the cell surface of HEK cells. This antibody, PS3B26, is a cell only binder and was culled from subsequent binding experiments. One of the positive binders, PS3B23 showed biphasic binding and did not fit well to the 1:1 binding model. Ten of the bispecific antibodies were positive binders to recombinant Chimp PSMA ECD by Proteon and their affinity was further profiled by BIACORE.

Binding to recombinant Chimp PSMA by Biacore. The Affinity measurements using Surface Plasmon Resonance (SPR) were performed using a Biacore 3000 optical biosensor (Biacore-GE Healthcare). A biosensor surface was prepared by coupling anti-Human IgG Fc (Jackson ImmunoResearch Laboratory, cat #109-005-098) to the carboxymethylated dextran surface of a CM-5 chip (Biacore, cat # BR-1000-12) using the manufacturer instructions for amine-coupling chemistry. Approximately 16,000 RU (response units) of anti-Human IgG Fc antibodies were immobilized in each of four flow cells. The kinetic experiments were performed at 25° C. in running buffer (DPBS+0.03% P20+100 g/ml BSA). Dilutions of antigen (recombinant Chimp PSMA, concentration either from 1.2-300 nM or 3.7-900 nM in a 3-fold serial dilution) were prepared in running buffer. About 100 RU of PSMA×CD3 bispecific mAbs were captured on flow cell 2 to 4 of the sensor chip. Flow cell 1 was used as reference surface. Capture of PSMA×CD3 bispecific mAb was followed by a 3 or 5-minute injection (association phase) of antigen (recombinant Chimp PSMA) at 50 μl/min, followed by 15 or 20 minutes of buffer flow (dissociation phase). The chip surface was regenerated by two 18-second injections of 100 mM Phosphoric acid (H₃PO₄, Sigma, cat #7961) at 50 μl/min.

The collected data were processed using BIAevaluation software (Biacore). First, double reference subtraction of the data was performed by subtracting the curves generated by buffer injection from the reference-subtracted curves for analyte injections. Then kinetic analysis of the data was performed using a Langmuir 1:1 binding model with global fit. The result for each PSMA×CD3 bispecific mAb was reported in the format of k_(a) (On-rate), k_(d) (Off-rate) and K_(D) (Equilibrium dissociation constant).

TABLE 19 Summary of kinetics and affinity for Bispecific mAbs binding to recombinant chimp PSMA ECD by Biacore ka (1/Ms) kd(1/s) KD (nM) PS3B19 6.24E+03 4.14E−04 66.3 PS3B20 7.82E+03 4.61E−04 59.1 PS3B21 1.10E+04 4.89E−04 44.4 PS3B22 1.66E+04 1.00E−03 60.4 PS3B23 1.34E+05 1.22E−03 9.1 PS3B24 2.35E+04 9.07E−04 38.6 PS3B25 6.59E+04 1.08E−03 16.4 PS3B27 8.90E+04 2.00E−03 22.4 PS3B28 6.20E+04 1.91E−03 30.8 PS3B29 2.74E+04 1.59E−03 58.1 PS3B30 3.48E+04 1.82E−03 52.3

All bispecific antibodies that bound to Chimp PSMA ECD by Proteon, also bound via Biacore. Binding affinities were somewhat weaker by Biacore.

Example 9. Evaluation of PSMA×CD3 Bispecific Abs in Functional Cell Killing Assay

T cell mediated cytotoxicity assays were used as a functional screen of bispecific antibody activity. Bispecifics were tested for the ability to lyse human PSMA overexpressing HEK cells, as well as the human prostate cancer cell line LNCAP. In addition, bispecifics were tested for the ability to kill cyno PSMA HEK cells to confirm species cross reactivity

A Chromium-51 release assay was used to measure cytotoxicity of individual bispecific antibodies. Cytotoxicity is measured by the amount of chromium release into the culture medium as a result of cell lysis in the presence of activated T-cells. The amount of release is compared to spontaneous release of chromium in target cells only and maximum release via total target cell lysis with Triton-X.

Human Pan T-cells (CD3+) from multiple donors were pre-activated overnight with OKT3 coated flasks (1 ug/ml) and IL-2 at 20 U/ml. T-cells were washed 2×. The target cell line was labelled for 1 hr with Chromium-51. T-cells and target cells were cultured at a 5:1 ratio for 18-24 hours before culture supernantant was harvested and analyzed. All points were run in triplicate and reported as a Cytoxoicity Mean of the triplicate and SEM. Dilutions of bispecific antibody from 10 ug/ml-0,00001 ug/ml were used.

FIG. 15A and FIG. 15B show T cell mediated killing for all PSMA×CD3 bispecific antibodies against hPSMA-HEK cells. For this experiment, the medium and high affinity CD3 arm bispecific antibodies were tested. Cell killing is evident for most of the high affinity CD3 arm molecules, with several of the bispecific antibodies showing killing at the lowest concentration tested. PS3B9 was the only medium affinity bispecific antibody that showed significant cell killing at lower concentrations. FIG. 16A and FIG. 16B show T cell mediated killing for several pairs of bispecific antibodies. From this data it is clear that the high affinity CD3 binding bispecific antibodies generate the most cell killing and these were the subject of further cell killing experiments.

FIG. 17 shows cell killing data for PSMA×CD3 bispecific antibodies with the high affinity CD3 arm generated against LNCAP cells. PS3B25, PS3B27, PS3B28, PS3B30, PS3B23 and PSB22 all had T cell redirection activity. PS3B29 showed activity on hPSMA-HEK cell at the highest concentrations but not on LNCAPs suggesting that because it is a weak PSMA binder, it is not able to bind well when PSMA expression levels are low. In contrast, PS3B25, PS3B27 and PS3B28 showed the highest LNCAP tumor cell lysis. A bispecific antibody PS3B38 consisting of PSMB125 and a null arm against RSV protein did not have cytolytic activity as expected due to inability to bind T cells.

To confirm cyno PSMA T cell redirection function Bispecific antibodies were tested for cyno PSMA-HEK cell lysis, shown on FIG. 18. All Bispecific antibodies tested were able to kill cells expressing cyno PSMA with PS3B27 and PS3B28 having the highest activity. From previous cell binding studies these anti-PSMA arms had higher affinity for cyno versus human PSMA and this observation was also reflected in cyno PSMA target cell killing. PS3B27 and PS3B30 were tested for killing of the parental HEK cell line and did not lyse the HEK parent cell line (data not shown).

The PSMA antibodies PSMB127 and PSMB130, which generate the PSMA×CD3 Bispecific antibodies PS3B27 and PS3B30, respectively, were selected for further analysis. PS3B27 and PS3B30 killed both human and cyno PSMA targets and had the high affinity CD3 arm. PS3B27 bound human PSMA expressing cell lines with substantially the same, EC₅₀'s of ˜14.6 nM and cyno of ˜9.9 nM. PS3B30 bound human PSMA with approximately 5-6 fold affinity difference, with EC₅₀'s of ˜8-8.5 nM and cyno PSMA at ˜37.8-57 nM. Although the difference between human and cyno binding may be greater than 5-fold, PS3B27 showed functional killing of both human and cyno targets.

The interactions of PS3B27 with recombinant Chimp PSMA ECD was repeated and interactions with recombinant Cyno PSMA ECD and Human PSMA ECD were studied by Surface Plasmon Resonance (SPR) using a ProteOn XPR36 system (BioRad) as described previously for recombinant chimp PSMA ECD. Summary of kinetics and affinity for binding to chimp, cyno and human PSMA ECD are shown in Table 20. This bispecific antibody binds all targets with substantially the same affinity.

TABLE 20 Summary of kinetics and affinity for PS3B25 and PS3B27 binding to recombinant target PSMA ECDs k_(a) (1/Ms) 10⁵ k_(d) (1/s) 10⁻⁰³ K_(D) (nM) target PS3B25 PS3B27 PS3B25 PS3B27 PS3B25 PS3B27 Chimp 2.81 ± 0.08 2.08 ± 0.38 0.99 ± 0.04 1.56 ± 0.37 3.54 ± 0.25 7.48 ± 0.97 Cyno 0.98 ± 0.04 1.59 ± 0.12 7.97 ± 0.34 1.10 ± 0.04 81.1 ± 3.3  7.00 ± 0.68 Human 1.88 ± 0.13 2.87 ± 0.36 1.01 ± 0.05 2.89 ± 0.70 5.38 ± 0.55 10.3 ± 3.2 

Evaluation of bispecific antibody, PS3B27 in a caspase assay. T-cell mediated killing of PS3B27 was measured using a second cell toxicity assay. The caspase cytotoxicity assay indirectly measures cell killing via cleavage of a fluorescent substrate by active caspase 3/7. Cleavage of the substrate results in a fluorescent DNA dye, with fluorescence restricted to the cell nucleus. Repeated fluorescence measurements are taken in each well throughout the course of the assay, using a motorized 1 OX objective, capable of precisely imaging well(s) at the same coordinates. Target cell populations are identified based on defined size restrictions and/or through the use of a secondary label.

Frozen Pan CD3+ T-cells (purchased from Biological Specialty Corporation, Colmar, Pa.) were isolated by negative selection from normal healthy donors. Prostate cancer cells expressing PSMA (LNCaP, C42) were cultured in RPMI 1640 with 10% HI FBS+supplements (purchased from Life Technologies).

T-cells and target cells were combined at an effector to target ratio (E:T) of 3:1 in Phenol Red free RPMI+10% FBS and supplements (Life Technologies), without selection reagents, and 0.6 uL of NucView caspase reagent (Essen Bioscience) was added to each mL of cells, per manufacturer guidelines. A total volume of 0.1 mL cells were added to appropriate wells of a clear, 96-well flat-bottom plate (BD Falcon). PS3B27 (CD3×PSMA), CD3B288 (CD3×Null) or PS3B46 (PSMAxNull) Bispecific antibodies were prepared at 2× final concentration in Phenol Red free RPMI, prepared as indicated above, and 0.1 mL of compounds were added to each well. After a 30 minute incubation at room temperature to minimize cell aggregation at the edge of wells, plates were transferred to the Zoom Incucyte instrument (Essen Bioscience). The Incucyte Instrument resides in a humidified incubator set at 37° C., 5% CO2.

Processing definitions on the Incucyte were designed for each cell line tested, per manufacture guidelines. Measurements were taken every six hours, until a plateau in the caspase signal was observed, and followed by three or more successive decreases from the maximum signal in the well(s) containing the highest concentration of the test compound(s).

After the assay was complete, each plate was analyzed using the appropriate processing definition. Raw fluorescent data was exported from the Incucyte Zoom software, and pasted into GraphPad Prism (GraphPad Software, Inc., La Jolla, Calif.). Caspase 3/7 activity was determined by calculating the area under the curve (AUC) for each well in GraphPad. AUC values were plotted as a function of Log 10 nM compound. The EC₅₀ for each dose curve, in nanomolar (nM), was reported following non-linear regression analysis (4 parameter fit, least ordinary squares). Each assay contained a minimum of three biologic replicates, and each cell line was tested with five healthy donors. Data were further analyzed by non-clinical statistics using a non-linear regression model. Examples of graphs are shown in FIG. 19A-C. Calculated results are found in Table 21.

TABLE 21 Summary of EC₅₀ Values for T-Cell Dependent Cytotoxicity Assay Donor LNCaP FGc C-42B M2550 0.27 nM 0.11 nM M5524 0.09 nM 0.06 nM M5772 0.04 nM 0.03 nM M7259 0.05 nM 0.06 nM M7444 0.09 nM 0.05 nM

Example 10. T-Cell Activation by PS3B27 in PSMA Positive Cell Lines

Purified Pan CD3+ T-cells were obtained from normal, healthy donors by Biological SpecialtyCorporation by negative selection of leukapheresed white blood cells, and stored frozen at −80° C. or in Liquid Nitrogen until ready for use. Naïve, unactivated T-cells were combined with target cells and CD3×PSMA bispecific antibodies or null controls (CD3×Null or PSMAxNull) at a 3:1 Effector:Target ratio. Following a 48-hour incubation, supernatants were analyzed for cytokine secretion by sandwich enzyme-linked immunosorbent assay (ELISA) (Meso Scale Discovery). Expression of the T-cell activation marker CD25 was measured by flow cytometry by staining T-cells for CD45, CD8, CD25, and a live/dead near-IR stain. Populations of CD8+/CD25+ were determined by first gating on a gross cell population (FSC-A vs. SSC-A) to exclude debris and cell aggregates. The cell gate subset was further narrowed for cells determined to be live, by exclusion of the live/dead stain. Live cells were then gated for CD45+/CD8+ cells. Finally, the CD8+/CD25+ positive subset was identified. The EC₅₀ of PS3B27 or control was derived by plotting the percentage of CD8+/CD25+ against Log 10 nM bispecific antibody or control, followed by a Non-linear regression (4 Parameter fit, least squares method) (FIG. 20). All data analysis was performed in GraphPad Prism.

Example 11. Anti-Tumor Efficacy of in Tumorigenesis Prevention of HEK293-PSMA Xenografts in PBMC-Humanized NSG Mice

All in vivo experiments were performed in accordance with The Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Janssen R & D, Spring House, Pa.

Efficacy of PS3B27 (PSMA×CD3 Bispecific antibody) was evaluated by prevention of tumorigenesis (prophylactic model) of HEK293-PSMA human xenografts using inoculated human donor peripheral blood mononuclear cells (PBMC) in male NSG mice (NOD.Cg-Prkdc^(scid)IL2rg^(tm/Wjl)/SzJ or NOD SCID Gamma, Jackson Laboratories, Bar Harbor, Me.). Mice were injected intravenously (iv) in the lateral tail vein with 1×10⁷ human PBMCs 7 days prior to tumor cell implantation. Mice were subsequently implanted subcutaneously (sc) with 1×10⁷ HEK293-PSMA cells in the right hind flank. Beginning on the day of tumor implantation PBS (phosphate buffered saline) control, PS3B27, CD3B288 (CD3×Null) or PS3B46 (PSMA×Null) were administered iv at 0.4 mg/kg q2d-q3d for a total of 5 doses on days 0, 3, 5, 7 and 10.

Tumor volume was calculated using the formula: Tumor Volume (mm³)=(a×b²/2); where ‘a’ represents the length, and ‘b’ the width of the tumor as determined by caliper measurements, and monitored twice weekly throughout the study. Percent tumor growth inhibition (TGI) was defined as the difference between mean tumor volumes of the treated and control (PBS) groups, calculated as TGI=[((TVc−TVt)/TVc)*100] where TVc is the mean tumor volume of a given control group and TVt is the mean tumor volume of the treated group. As defined by NCI criteria, ≥60% TGI is considered biologically significant (Johnson, et al (2001) Br J Cancer 84(10) 1424-31). Animals were removed from studies when a maximum tumor volume of 1500 mm³ was reached.

Engraftment of human PBMC eventually leads to graft-versus-host disease (GvHD) in the mice, where the engrafted donor T cells become activated and infiltrate the host tissues, leading to body weight loss, organ failure, and inevitably, death. To monitor the onset and severity of GvHD, body weight was recorded twice weekly and expressed in grams (g). Percent body weight change was calculated using the formula: Body weight change=[((B_(t)−B₀)/B₀)*100] where B_(t) is the body weight on a given day of study and B₀ is the body weight at the initiation of treatment. Animals with sustained body weight loss greater than 20% of the initial body weight were considered moribund and removed from the study.

Statistical significance was evaluated using a 1-way ANOVA with multiple comparisons using Dunnett's multiple comparisons test using Graph Pad Prism software (version 6). PS3B27 treatment effectively delayed HEK293-PSMA tumorigenesis and tumor growth (FIG. 21). Small but palpable HEK293-PSMA tumors were detectable in seven of eight mice in the PBS treated group on study day 16 (6 days post last therapeutic treatment), whereas only one mouse out of eight in the PS3B27 treated group had a tumor. Five out of eight mice had palpable tumors in the CD3B288 treatment group and two out of eight mice had small tumors in the PS3B46 group. Tumor growth inhibition was assessed 27 days following cessation of treatment (day 37 post-tumor implantation), when each group had a minimum of 7 animals. Tumor growth in the PSMA×CD3 bispecific antibody (PS3B27) treated group was inhibited by 90% as compared to PBS-treated controls (n=8/group, p<0.001). The PSMA×Null bispecific antibody (PS3B46) also inhibited tumorigenesis and growth in a statistically significant fashion (TGI=42%, n=7) vs. PBS control, (p<0.05), although it was not considered to be a biologically significant effect based on NCI criteria [1].

Animal groups receiving PBMCs eventually succumb to progressive GvHD, however body weight loss was slight in the current study. No significant difference was observed between mean body weights of animals treated with 0.4 mg/kg PS3B27vs PBS as shown in FIG. 22 up to day 37 post-tumor implant (p>0.05). Therefore PS3B27-mediated T cell redirection did not further contribute towards GvHD-related body weight loss.

Despite minor weight loss in the current study, sporadic GvHD-related deaths were noted. One mouse in the PSMA×Null bispecific antibody PS3B46 group was euthanized due to excessive GVHD-related (>20%) body weight loss on day 30-post tumor implant. By day 42 post-tumor implant, additional GvHD-related deaths were noted in the PBS (n=1), and PSMA×Null bispecific antibody PS3B46 groups (n=2), and several additional mice were removed from the study due to reaching the 1500 mm³ tumor volume endpoint, at which time the entire study was terminated.

Example 12. Efficacy of PS3B27 in Tumorigenesis Prevention of Admixture HEK293-PSMA/T Cell Xenografts in Male CD1 Nude Mice

Efficacy of PS3B27 was evaluated in an admixture xenograft model where human CD3+ pan T cells and tumor cells were co-injected into male CD1 nude mice (NU-Foxn1nu, Charles River Laboratories, Wilmington, Mass.).

Human PSMA x human CD3 bispecific antibody PS3B27, or control bispecific antibodies were administered iv every 2-3 days (q2d or q3d) for a total of 5 doses as indicated. Mice were monitored (body weight and tumor caliper measurement) twice weekly throughout the studies. Drug doses expressed as μg/animal were converted to mg/kg based on a 25 g body weight (example: 10 μg/animal=0.4 mg/kg). Drug doses administered as mg/kg, were dosed 10 mL/kg based on body weight (example: 25 g mouse=0.25 mL).

Tumor volume was calculated using the formula: Tumor Volume (mm3)=(a×b2/2); where ‘a’ represents the length, and ‘b’ the width of the tumor as determined by caliper measurements], and monitored twice weekly throughout the study. Percent tumor growth inhibition (TGI) was defined as the difference between mean tumor volumes of the treated and control (PBS) groups, calculated as TGI=[((TVc−TVt)/TVc)*100] where TVc is the mean tumor volume of a given control group and TVt is the mean tumor volume of the treated group. As defined by NCI criteria, ≥60% TGI is considered biologically significant [1]. Animals were removed from studies when a maximum tumor volume of 1500 mm3 was reached.

The tolerability of PS3B27 could not be assessed with respect to CD3 binding in host tissues due to lack of cross-reactivity of the CD3 arm to corresponding mouse antigens. The T cell injected with the tumor cells do however express human CD3 and can bind PS3B27 and CD3X Null controls. Percent body weight change was calculated using the formula: Body weight change=[((Bt B0)/B0)*100] where Bt is the body weight on a given day of study and B0 is the body weight at the initiation of treatment.

Statistical significance was evaluated using a 1-way ANOVA with multiple comparisons using Dunnett's multiple comparisons test using Graph Pad Prism software (version 6).

Efficacy of PS3B27 was evaluated by prevention of tumorigenesis of admixture xenografts containing HEK293-PSMA cells and activated and expanded CD3 positive pan T-cells in a 1:5 effector to target ratio in male CD1 nude mice (ELN ref: CD3-PSMA-2013-00003). T-cells were activated and expanded in vitro using the T-cell activation/expansion kit in IL-2 containing media (Miltenyi Biotech, Auburn, Calif., catalog #130-091-441, 130-097-743) for 12 days. Mice were implanted sc with an admixture of 5×106 HEK293-PSMA cells and 1×106 activated and expanded T-cells per mouse in 50% Cultrex (Trevigen, Gaithersburg, Md., catalog #3433-005-01) and 50% serum-free RPMI 1640 media in the right hind flank. Beginning on the same day as tumor implantation, PBS, PS3B27 at 0.005-0.5 mg/kg, CD3B288 (CD3×Null bispecific antibody) 0.5 mg/kg or PS3B46 (PSMA×Null bispecific antibody) 0.5 mg/kg were administered iv, by body weight, q2d-q3d for a total of 5 doses on days 0, 2, 4, 7 and 9. (n=10/group). Treatment with PS3B27 was also evaluated with ip administration (data not shown). One animal was removed each on days 46 and 49 in the PBS control group for excessive tumor burden. Tumor volume data was plotted up to day 64 after which half of the control animals were removed from study due to excessive tumor volume.

As shown in FIG. 23, tumorigenesis and growth were evaluated for 55 days following cessation of treatment (up to day 64). Treatment with PS3B27 significantly inhibited tumorigenesis and delayed growth compared to PBS control at all doses (0.005, 0.05 or 0.5 mg/kg) resulting in TGI of 73%, 81% and 82%, respectively (p<0.001, P<0.0001, P<0.001, respectively) on day 64. Treatment of PS3B27 by ip administration showed similar efficacy as iv administration (data not shown). Animals treated with CD3B288 (CD3×Null bispecific antibody) or PS3B46 (PSMA×Null Bispecific antibody) showed some anti-tumor activity with 51% and 38% TGL respectively on day 64 (p<0.05, p=ns, respectively), however this is not considered biologically significant based on the NCI criteria of 60% TGI, demonstrating the requirement for both CD3 and PSMA binding of the bispecific antibody to achieve efficacy.

There was no body weight loss over the course of the study, however, animals treated with PS3B27 at 0.5 and 0.005 mg/kg did have significantly less increase in body weight compared to PBS (p<0.001, p<0.0001, respectively, FIG. 24), however this could be due to a lower tumor burden in these animals.

Example 13. Crystal Structure of Human PSMA ECD Bound to Anti-PSMA Fab Arm of Bispecific Antibody PS3B27

PSMA is a homodimeric protein expressed on the cell surface. PSMA is a type II integral glycoprotein of 750 residues per monomer, comprised of a large ECD domain (705 residues) with peptidase activity, a single pass TM domain, and a short 19 residue intracellular domain. The crystal structure of the extracellular region (ECD) of human PSMA bound to the anti-PSMA Fab arm of bispecific antibody PS3B27 was determined to 3.15 Å resolution to better understand the combining site between PSMA and the antibody.

The extracellular region of human PSMA (residues 44-750) was expressed in High Five™ insect cells with an N-terminal gp67 signal peptide followed by a cleavable hexahistidine tag (SEQ ID NO: 158). The secreted protein was purified from supernatant by a three-step procedure comprising of an initial Ni²⁺-NTA affinity capture, TEV-mediated cleavage of the histidine tag followed by an inverse affinity chromatography step, and a final size-exclusion chromatography step. Purified PSMA-ECD was flash-frozen in liquid nitrogen and stored at −80° C. in 10 mM HEPES pH 7.4, 150 mM NaCl, 2 mM CaCl₂), 0.1 mM ZnCl₂

The Fab of PSMB83, which is the parental anti-PSMA Fab arm in bispecific antibody PS3B27, was expressed in HEK293 Expi cells with a hexahistidine tag (SEQ ID NO: 158) and purified using affinity (HisTrap, GE Healthcare) and size-exclusion chromatography (SEC-300, Phenomenex Yarra). The Fab was stored at 4° C. in 50 mM NaCl, 20 mM Tris pH 7.4

The human PSMA ECD/PSMB83 Fab complex was prepared by a three-step procedure. First, the Fab was buffer exchanged into 20 mM MES pH 6.0, 150 mM NaCl. Then, the Fab and PSMA were mixed (1.5 molar excess Fab over PSMA monomer) and incubated overnight at 4° C. while dialyzing into 20 mM MES pH 6.0. Finally, the complex was bound to a monoS 5/50 column in 20 mM MES pH 6.0 and eluted with a NaCl gradient.

Crystals suitable for X-ray diffraction were obtained using the sitting drop vapor-diffusion method at 20° C. and a Mosquito LCP robot (TTP Labtech). Crystals of PSMB83 Fab bound to human PSMA ECD were grown from 18% PEG 3 kDa, 0.2 M (NH₄)₂SO₄, 0.1 M Tris pH 8.5 with micro-seeds and the PSMA/Fab complex initially at 7.3 mg/mL. Crystals of free PSMB83 Fab were obtained from 25% PEG 3 kDa, 0.2 M LiCl, 0.1 M acetate pH 4.5 with the Fab initially at 8.8 mg/mL.

The structures were solved by molecular replacement (MR) with Phaser (Phaser Crystallographic Software, University of Cambridge). The MR search model for the PSMB83 Fab structure was PDB code 4M60. The PSMA/Fab complex structure was solved using the crystal structures of PSMA (PDB code: 2C6G) and PSMB83 Fab (structure at 1.93 Å resolution; data not shown) as MR search models. The structures were refined with PHENIX (Adams, et al, 2004) and model adjustments were carried out using COOT (Emsley and Cowtan, 2004). All other crystallographic calculations were performed with the CCP4 suite of programs (Collaborative Computational Project Number 4, 1994). All molecular graphics were generated with PyMol (PyMOL Molecular Graphics System, Version 1.4.1, Schrödinger, LLC.) and complementarity determining regions (CDRs) were determined using the Kabat definition.

The PSMA/Fab structure includes Fab light chain residues 1-211, Fab heavy chain residues 1-224 (except for residues 138-146, which are disordered) and PSMA residues 56-750, which corresponds to the protease (residues 56-116 and 352-590), apical (residues 117-351) and helical (residues 591-750) domains, and seven of ten possible N-linked glycans (in Asn-76, -121, -140, -195, -459, -476, and -638) per PSMA dimer subunit. The PSMA active site is located at the interface between the three domains and it contains two zinc atoms coordinated by histidine (H377 and H553) and glutamate/aspartate (D387, catalytic E424, E425, and D453) residues and a water molecule. The crystal asymmetric unit contains one PSMA dimer with each subunit bound in a similar manner to a PSMB83 Fab. The Fab/PSMA combining site is well defined by the electron density map, which allows reliable positioning of the binding residues. The Fab and PSMA molecules are numbered sequentially in FIGS. 25-30

The PSMB83 epitope, paratope and interactions. PSMB83, which is the parental anti-PSMA Fab arm in bispecific antibody PS3B27, recognizes a conformational and discontinuous epitope in the apical domain of PSMA (FIG. 25). The PSMA surface area buried by the Fab is around 700 Å². Specifically, the PSMB83 epitope residues are 1138, F235, P237, G238, D244, Y299, Y300, Q303, K304, E307, and K324-P326. Helix α7 (residues Y299-E307) is a prevalent region of the epitope and binds across the Fab heavy and light chain CDRs. At one end of the helix, Y299 and Y300 form an aromatic cluster with Fab residues Y57^(H), W94^(L) and PSMA residues F235 and P237, while E307, at the other helix end, forms a salt bridge with R91^(L) and hydrogen bonds Y32^(L). FIGS. 26 and 27 show the main interactions of PSMA with the PSMB83 light and heavy chains. The PSMB83 epitope residues are conserved between human and cynomolgus monkey (FIG. 28) and the bispecific antibody PS3B27 was demonstrated to bind with similar affinity to human and cyno PSMA. In contrast, the human to mouse G238A and, especially. Y300D epitope mutations are expected to lower PSMB83 binding affinity to mouse PSMA in comparison to human. The Y300D mutation disrupts a hydrogen bond contact with N59^(H) and a 7t stacking interaction with W94^(L). The PSMB83 paratope is composed of residues from all CDRs except CDR-L2 and CDR-H1 (FIG. 29). Specifically, the paratope residues are light chain S30^(L), Y32^(L), R91^(L), S92^(L), W94^(L), and heavy chain G56H-N59^(H), K65^(H), G66^(H), Y101^(H), V107^(H), and D109^(H). FIG. 30 shows the interaction contacts between PSMA and PSMB83. The accessible location of the epitope facilitates binding of the PSMB83 Fab arm in the PS3B27 bispecific antibody to membrane-bound PSMA, while the other Fab arm is still bound to CD3 in the T-cell membrane. PSMB83 is not expected to inhibit PSMA enzymatic activity since the antibody binds away from the active site and does not cause any significant structural changes in PSMA that could affect enzymatic function, such as loop movements that close the active site or displacement of catalytic residues (RMSD of 0.3 Å for Cα superposition of PSMA molecules in Fab bound and unbound (Barinka et al, 2007) structures)

Example 14. Anti-PSMA Affinity Maturation

Affinity maturation was performed on anti-PSMA Fab phage clones from two PSMA affinity maturation libraries to identify an antibody with increased binding affinity compared to the parental PSMB127 (fab ID=PSMB83). Two libraries were generated for affinity maturation of PSMB127. In the first library heavy chain CDR1 and CDR2 were randomized according to the design in Table 22 (PH9H9L1). The H-CDR3 fragment was PCR amplified from pDR000024032 and digested with SacII+XhoI. This fragment was cloned into the PH9H9L1/PH9L3 library. This was transformed into E. coli MC1061F′ cells and phage was generated displaying this Fab library. In the second library light chain CDRs were randomized according to the design in Table 25 (PH9L3L3). The heavy chain from PSMB83 (PSMH360) was PCR amplified and digested with NcoI+XhoI. This fragment was cloned into the PH9L3L3 library DNA (ELN: De Novo 2010 phage library SRI-021). This was transformed into E. coli MC1061F′ cells and phage was generated displaying this Fab library.

TABLE 22 PH9H9L1 Library design Position Parent AA Library AA 30 S D, K, S 31 S D, N, S, T 32 Y A, D, S, Y 33 A A, D, G, S, W, Y 35 S H, N, S 50 A A, E, L, N, R, T, W, Y 52 S A, D, L, N, R, S 54 S A, E, N, S, Y 57 S D, N, R, S, T, Y 59 Y E, G, N, Q, R, Y

TABLE 23 PH9L3L3 Library design Position Parent AA Library AA 30 S D, N, R, S 31 S N, S, T 32 Y D, N, R, S, Y 49 Y E, H, K, Y 50 D D, G, S, W, Y 53 N D, N, S, T, Y 91 R A, D, E, G, H, N, R, S, W, Y 92 S A, D, E, G, H, N, R, S, W, Y 93 N A, D, E, G, H, N, R, S, W, Y 94 W A, D, E, G, H, N, R, S, W, Y 96 L F, I, L, N, R, W, Y

A solution panning of the PSMA affinity maturation Fab-pIX libraries was performed against biotinylated human PSMA ECD for three rounds. The phage-bound antigen was captured on neutravidin beads (GE HealthCare Life Science Cat #78152104011150) according to the manufacturer's protocol, followed by extensive washes in 1×PBST (0.05% tween 20) and an hour-long incubation with unlabeled PSMA ECD in 500-fold molar excess of the biotinylated antigen. This panning yielded the clones, PSMXP46R3_59H09, PSMXP46R3_59H06, PSMXP46R3_59E03, PSMXP46R3_59C09, PSMXP46R3_59H01, PSMXP46R3_59F11, and PSMXP46R3_59F07.

To determine the expression level of the anti-PSMA fab clones, 96 well Maxisorb plates were coated overnight at 4 C with anti-human Fd IgG, washed, and blocked with 3% milk-PBS-0.05% Tween for 1 hour. The phage supernatant samples were serially diluted 2-fold for 11 dilutions in blocking buffer with the final well blank. 100 ul of these solutions were captured on the coated plates for 1 hour. The plates were washed and 100 ul of anti-F(ab′)2-HRP antibody was added for 1 hour. Plates were washed and developed with 100 ul of peroxidase reagent and luminescence was read on the Envision (FIG. 31).

To determine the binding of the anti-PSMA fab clones to human and cynomolgus recombinant protein, 96 well Maxisorb plates were coated with 100 ul of 5 ug/ml neutravidin overnight at 4 C. The plates were washed and blocked with 3% milk-PBS-0.05% Tween for 1 hour. Recombinant biotinylated human and cynomolgus PSMA proteins were captured at 2.5 ug/ml for 1 hour at room temperature. The plates were washed and 100 ul of 2-fold serially diluted fab supernatant was capture for 1 hour at RT. The plate was washed and then there was a 2.5-hour incubation with 200 ul 0.3% milk in PBST to wash away some of the weak affinity fabs. Then there was another 30 minute incubation with fresh 200 ul 0.3% milk in PBST to remove more weak affinity fabs. The plates were washed and 100 ul of anti-F(ab′)2-HRP antibody was added for 1 hour. Plates were washed and developed with 100 ul of peroxidase reagent and luminescence was read on the Envision (FIG. 32 and FIG. 33). FIG. 31 demonstrates that the protein expression of the parental Fab and affinity matured Fabs were similar. The y axis values represent the luminescence of the detection reagent which equates to the abundance of fab protein over the dilution curve; the higher the luminescence reading, the more protein in the well which decreased with successive two-fold dilutions. There was more protein in the wells with affinity mature fabs but the increase over the parental is at most five times greater as demonstrated by the EC₅₀ values (which is the concentration of protein that gives half of the maximal response). These data demonstrate the difference in PSMA binding profiles in FIG. 32 and FIG. 33 is not due to a difference in Fab concentration.

FIG. 32 demonstrates improved binding of the affinity matured Fabs to the human recombinant antigen over the parental anti-PSMA Fab (PSMB83). Again, the y axis of the graph represents luminescence values. In this case the larger the value means more Fab bound to the human PSMA protein. This is a measure of binding as increased concentrations of Fab (along the x axis) generate higher luminescence values. There was negligible binding of the parental Fab under these conditions as demonstrated by the absence of signal even at high concentrations (open circles along the x axis). Binding of the affinity matured fabs was observed over the concentrations tested which equates to stronger binding capacity to the human PSMA protein. Given that parental Fab binding to human PSMA protein was zero, no EC₅₀ could be generated.

FIG. 33 demonstrates improved binding of the affinity matured Fabs to the cynomolgus recombinant antigen over the parental anti-PSMA Fab (PSMB83). Again, the y axis of the graph represents luminescence values. In this case the larger the value means more Fab bound to the cynomolgus PSMA protein. This is a measure of binding as increased concentrations of Fab (along the x axis) generate higher luminescence values. There was negligible binding of the parental Fab under these conditions as demonstrated by the absence of signal even at high concentrations (open circles along the x axis). Binding of the affinity matured Fabs was observed over the concentrations tested which equates to stronger binding capacity to the human PSMA protein. Given that parental Fab binding to human PSMA protein was zero, no EC₅₀ could be generated for direct comparison.

Overall, the phages' Fab binding profiles demonstrate improved binding to the human and cynomolgus recombinant antigen over the parental anti-PSMA mAb (PSMB127). This improvement is not a result of differences in Fab expression profiles, as demonstrated by FIG. 34 and FIG. 35, showing binding of affinity mature Fabs normalized to Fab expression levels. The top five Fab candidates identified from the ELISA screen were produced in monoclonal antibody format on IgG4 PAA. Table 24 lists the subsequent Mab identifiers and Tables 25 and 26 provide sequence information.

TABLE 24 Top five affinity mature antibodies identified based on the ELISA Well ID HC SEQ ID LC SEQ ID MAB protein IDs PSMXP46R3_59C09 PSMH859 PSML160 PSMB346 PSMXP46R3_59E03 PSMH859 PSML159 PSMB345 PSMXP46R3_59F07 PSMH862 PSML158 PSMB349 PSMXP46R3_59H01 PSMH860 PH9L3 PSMB347 PSMXP46R3_59H06 PSMH859 PH9L3 PSMB344

TABLE 25 VH and VL sequences of top five PSMA Fab candidates MAB ID VH Amino acid sequence SEQ ID NO VL Amino Acid Sequence SEQ ID NO PSMB344 EVQLLESGGGLVQPGGSLRLSCAASG 138 EIVLTQSPATLSLSPGE  67 FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSSYLA GSGGYTNYADSMKGRFTISRDNSKNT WYQQKPGQAPRLLIYDA LYLQMNSLRAEDTAVYYCARDSYDSS SNRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSS TDFTLTISSLEPEDFAV YYCQQRSNWPLTFGQGT KVEIK PSMB345 EVQLLESGGGLVQPGGSLRLSCAASG 138 EIVLTQSPATLSLSPGE 142  FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSNYLA GSGGYTNYADSMKGRFTISRDNSKNT WYQQKPGQAPRLLIHDA LYLQMNSLRAEDTAVYYCARDSYDSS SNRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSS TDFTLTISSLEPEDFAV YYCQQRRNWPLTFGQGT KVEIK PSMB346 EVQLLESGGGLVQPGGSLRLSCAASG 138 EIVLTQSPATLSLSPGE 143 FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSSYLA GSGGYTNYADSMKGRFTISRDNSKNT WYQQKPGQAPRLLIYDA LYLQMNSLRAEDTAVYYCARDSYDSS SYRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSS TDFTLTISSLEPEDFAV YYCQQRRNWPLTFGQGT KVEIK PSMB347 EVQLLESGGGLVQPGGSLRLSCAASG 139 EIVLTQSPATLSLSPGE  67 FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSSYLA GSGGYTNYADSMKSRFTISRDNSKNT WYQQKPGQAPRLLIYDA LYLQMNSLRAEDTAVYYCARDSYDSS SNRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSS TDFTLTISSLEPEDFAV YYCQQRSNWPLTFGQGT KVEIK PSMB349 EVQLLESGGGLVQPGGSLRLSCAASG 140 EIVLTQSPATLSLSPGE 144 FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSSYLA GSGGYTNYADSLKGRFTISRDNSKNT WYQQKPGQAPRLLIYDA LYLQMNSLRAEDTAVYYCARDSYDSS SNRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSS TDFTLTISSLEPEDFAV YYCQQRGNWPLTFGQGT KVEIK

TABLE 26 Heavy Chain and Light Chain sequences of top five PSMA candidates in monoclonal antibody format on IgG4 PAA Heavy Chain Amino acid SEQ ID Light Chain Amino Acid SEQ ID mAb ID sequence NO Sequence NO PSMB344 EVQLLESGGGLVQPGGSLRLSCAA 145 EIVLTQSPATLSLSPGERATLS  89 SGFTFKSDAMHWVRQAPGKGLEWV CRASQSVSSYLAWYQQKPGQAP SEISGSGGYTNYADSMKGRFTISR RLLIYDASNRATGIPARFSGSG DNSKNTLYLQMNSLRAEDTAVYYC SGTDFTLTISSLEPEDFAVYYC ARDSYDSSLYVGDYFDYWGQGTLV QQRSNWPLTFGQGTKVEIKRTV TVSSASTKGPSVFPLAPCSRSTSE AAPSVFIFPPSDEQLKSGTASV STAALGCLVKDYFPEPVTVSWNSG VCLLNNFYPREAKVQWKVDNAL ALTSGVHTFPAVLQSSGLYSLSSV QSGNSQESVTEQDSKDSTYSLS VTVPSSSLGTKTYTCNVDHKPSNT STLTLSKADYEKHKVYACEVTH KVDKRVESKYGPPCPPCPAPEAAG QGLSSPVTKSFNRGEC GPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQE EMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK PSMB345 EVQLLESGGGLVQPGGSLRLSCAA 145 EIVLTQSPATLSLSPGERATLS 148 SGFTFKSDAMHWVRQAPGKGLEWV CRASQSVSNYLAWYQQKPGQAP SEISGSGGYTNYADSMKGRFTISR RLLIHDASNRATGIPARFSGSG DNSKNTLYLQKNSLRAEDTAVYYC SGTDFTLTISSLEPEDFAVYYC ARDSYDSSLYVGDYFDYWGQGTLV QQRRNWPLTFGQGTKVEIKRTV TVSSASTKGPSVFPLAPCSRSTSE AAPSVFIFPPSDEQLKSGTASV STAALGCLVKDYFPEPVTVSWNSG VCLLNNFYPREAKVQWKVDNAL ALTSGVHTFPAVLQSSGLYSLSSV QSGNSQESVTEQDSKDSTYSLS VTVPSSSLGTKTYTCNVDHKPSNT STLTLSKADYEKHKVYACEVTH KVDKRVESKYGPPCPPCPAPEAAG QGLSSPVTKSFNRGEC GPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQE EMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK PSMB346 EVQLLESGGGLVQPGGSLRLSC 145 EIVLTQSPATLSLSPGERAT 149 AASGFTFKSDAMHWVRQAPGKG LSCRASQSVSSYLAWYQQKP LEWVSEISGSGGYTNYADSMKG GQAPRLLIYDASYRATGIPA RFTISRDNSKNTLYLQMNSLRA RFSGSGSGTDFTLTISSLEP EDTAVYYCARDSYDSSLYVGDY EDFAVYYCQQRRNWPLTFGQ FDYWGQGTLVTVSSASTKGPSV GTKVEIKRTVAAPSVFIFPP FPLAPCSRSTSESTAALGCLVK SDEQLKSGTASVVCLLNNFY DYFPEPVTVSWNSGALTSGVHT PREAKVQWKVDNALQSGNSQ FPAVLQSSGLYSLSSVVTVPSS ESVTEQDSKDSTYSLSSTLT SLGTKTYTCNVDHKPSNTKVDK LSKADYEKHKVYACEVTHQG RVESKYGPPCPPCPAPEAAGGP LSSPVTKSFNRGEC SVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGV EVHNAKTKPRESQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQFENNYK TTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK PSMB347 EVQLLESGGGLVQPGGSLRLSC 146 EIVLTQSPATLSLSPGERAT  89 AASGFTFKSDAMHWVRQAPGKG LSCRASQSVSSYLAWYQQKP LEWVSEISGSGGYTNYADSMKS GQAPRLLIYDASNRATGIPA RFTISRDNSKNTLYLQMNSLRA RFSGSGSGTDFTLTISSLEP EDTAVYYCARDSYDSSLYVGDY EDFAVYYCQQRSNWPLTFGQ FDYWGQGTLVTVSSASTKGPSV GTKVEIKRTVAAPSVFIFPP FPLAPCSRSTSESTAALGCLVK SDEQLKSGTASVVCLLNNFY DYFPEPVTVSWNSGALTSGVHT PREAKVQWKVDNALQSGNSQ FPAVLQSSGLYSLSSVVTVPSS ESVTEQDSKDSTYSLSSTLT SLGTKTYTCNVDHKPSNTKVDK LSKADYEKHKVYACEVTHQG RVESKYGPPCPPCPAPEAAGGP LSSPVTKSFNRGEC SVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGV EVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGK5YKCKVSNK GLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK PSMB349 EVQLLESGGGLVQPGGSLRLSC 147 EIVLTQSPATLSLSPGERAT 150 AASGFTFKSDAMHWVRQAPGKG LSCRASQSVSSYLAWYQQK LEWVSEISGSGGYTNYADSLKG GQAPRLLIYDASNRATGIPA RFTISRDNSKNTLYLQMNSLRA RFSGSGSGTDFTLTISSLEP EDTAVYYCARDSYDSSLYVGDY EDFAVYYCQQRGNWPLTFGQ FDYWGQGTLVTVSSASTKGPSV GTKVEIKRTVAAPSVFIFPP FPLAPCSRSTSESTAALGCLVK SDEQLKSGTASVVCLLNNFY DYFPEPVTVSWNSGALTSGVHT PREAKVQWKVDNALQSGNSQ FPAVLQSSGLYSLSSVVTVPSS ESVTEQDSKDSTYSLSSTLT SLGTKTYTCNVDHKPSNTKVDK LSKADYEKHKVYACEVTHQG RVESKYGPPCPPCPAPEAAGGP LSSPVTKSFNRGEC SVFLFPPKPKDTLMXSRTPEVT CVVVDVSQEDPEVQFNWYVDGV EVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK

The 3 different HC and 4 different LC were combined in a matrix format to expand the diversity of hits (Table 26). Given that the methionine in the CDR2 of PSMH860 is a posttranslational risk, a new sequence was generated with M64L and identified as PSMH865. PSMH865 was paired with PSML160 to generate Mab PSMB365. Tables 27 and 28 provide sequence information.

Table 26: Matrix format of the 3 heavy chains and 4 light chains combined

TABLE 27 VH and VL sequences of matrix recombined PSMA hits SEQ ID SEQ ID MAB ID VH Amino acid sequence NO VL Amino Acid Sequence NO PSMB358 EVQLLESGGGLVQPGGSLRLSCAASG 140 EIVLTQSPATLSLSPGE  67 FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSSYLA GSGGYTNYADSLKGRFTISRDNSKNT WYQQKPGQAPRLLIYDA LYLQMNSLRAEDTAVYYCARDSYDSS SNRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSS TDFTLTISSLEPEDFAV YYCQQRSNWPLTFGQGT KVEIK PSMB359 EVQLLESGGGLVQPGGSLRLSCAASG 140 EIVLTQSPATLSLSPGE 142 FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSNYLA GSGGYTNYADSLKGRFTISRDNSKNT WYQQKPGQAPRLLIHDA LYLQMNSLRAEDTAVYYCARDSYDSS SNRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSS TDFTLTISSLEPEDFAV YYCQQRRNWPLTFGQGT KVEIK PSMB360 EVQLLESGGGLVQPGGSLRLSCAASG 140 EIVLTQSPATLSLSPGE 143 FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSSYLA GSGGYTNYADSLKGRFTISRDNSKNT WYQQKPGQAPRLLIYDA LYLQMNSLRAEDTAVYYCARDSYDSS SYRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSS TDFTLTISSLEPEDFAV YYCQQRRNWPLTFGQGT KVEIK PSMB361 EVQLLESGGGLVQPGGSLRLSCAASG 139 EIVLTQSPATLSLSPGE 144 FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSSYLA GSGGYTNYADSMKSRFTISRDNSKNT WYQQKPGQAPRLLIYDA LYLQMNSLRAEDTAVYYCARDSYDSS SNRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSS TDFTLTISSLEPEDFAV YYCQQRGNWPLTFGQGT KVEIK PSMB362 EVQLLESGGGLVQPGGSLRLSCAASG 139 EIVLTQSPATLSLSPGE 142  FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSNYLA  GSGGYTNYADSMKSRFTISRDNSKNT WYQQKPGQAPRLLIHDA LYLQMNSLRAEDTAVYYCARDSYDSS SNRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSS TDFTLTISSLEPEDFAV YYCQQRRNWPLTFGQGT KVEIK PSMB363 EVQLLESGGGLVQPGGSLRLSCAASG 139 EIVLTQSPATLSLSPGE 143 FTFKSDAMHWVRQAPGKGLEWVSEIS  RATLSCRASQSVSSYLA GSGGYTNYADSMKSRFTISRDNSKNT  WYQQKPGQAPRLLIYDA LYLQMNSLRAEDTAVYYCARDSYDSS  SYRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSS TDFTLTISSLEPEDFAV  YYCQQRRNWPLTFGQGT KVEIK PSMB365 EVQLLESGGGLVQPGGSLRLSCAASG 141 EIVLTQSPATLSLSPGE 143 FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSSYLA GSGGYTNYADSLKSRFTISRDNSKNT WYQQKPGQAPRLLIYDA LYLQMNSLRAEDTAVYYCARDSYDSS SYRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSS  TDFTLTISSLEPEDFAV YYCQQRRNWPLTFGQGT KVEIK

TABLE 28 Heavy Chain and Light Chain sequences of matrix recombined PSMA hits Heavy Chain SEQ ID Light Chain SEQ ID MAB ID Amino acid sequence NO Amino Acid Sequence NO PSMB358 EVQLLESGGGLVQPGGSLRLSCAASG 147 EIVLTQSPATLSLSPGE  89 FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSSYLA GSGGYTNYADSLKGRFTISRDNSKNT WYQQKPGQAPRLLIYDA LYLQMNSLRAEDTAVYYCARDSYDSS SNRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSSASTKGP TDFTLTISSLEPEDFAV SVFPLAPCSRSTSESTAALGCLVKDY YYCQQRSNWPLTFGQGT FPEPVTVSWNSGALTSGVHTFPAVLQ KVEIKRTVAAPSVFIFP SSGLYSLSSVVTVPSSSLGTKTYTCN PSDEQLKSGTASVVCLL VDHKPSNTKVDKRVESKYGPPCPPCP NNFYPREAKVQWKVDNA APEAAGGPSVFLFPPKPKDTLMISRT LQSGNSQESVTEQDSKD PEVTCVVVDVSQEDPEVQFNWYVDGV STYSLSSTLTLSKADYE EVHNAKTKPREEQFNSTYRVVSVLTV KHKVYACEVTHQGLSSP LHQDWLNGKEYKCKVSNKGLPSSIEK VTKSFNRGEC TISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK PSMB359 EVQLLESGGGLVQPGGSLRLSCAASG 147 EIVLTQSPATLSLSPGE 148 FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSNYLA GSGGYTNYADSLKGRFTISRDNSKNT WYQQKPGQAPRLLIHDA LYLQMNSLRAEDTAVYYCARDSYDSS SNRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSSASTKGP TDFTLTISSLEPEDFAV SVFPLAPCSRSTSESTAALGCLVKDY YYCQQRRNWPLTFGQGT FPEPVTVSWNSGALTSGVHTFPAVLQ KVEIKRTVAAPSVFIFP SSGLYSLSSVVTVPSSSLGTKTYTCN PSDEQLKSGTASVVCLL VDHKPSNTKVDKRVESKYGPPCPPCP NNFYPREAKVQWKVDNA APEAAGGPSVFLFPPKPKDTLMISRT LQSGNSQESVTEQDSKD PEVTCVVVDVSQEDPEVQFNWYVDGV STYSLSSTLTLSKADYE  EVHNAKTKPREEQFNSTYRVVSVLTV KHKVYACEVTHQGLSSP LHQDWLNGKEYKCKVSNKGLPSSIEK VTKSFNRGEC TISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK PSMB360 EVQLLESGGGLVQPGGSLRLSCAASG 147 EIVLTQSPATLSLSPGE 149 FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSSYLA GSGGYTNYADSLKGRFTISRDNSKNT WYQQKPGQAPRLLIYDA LYLQMNSLRAEDTAVYYCARDSYDSS SYRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSSASTKGP TDFTLTISSLEPEDFAV SVFPLAPCSRSTSESTAALGCLVKDY YYCQQRRNWPLTFGQGT FPEPVTVSWNSGALTSGVHTFPAVLQ KVEIKRTVAAPSVFIFP SSGLYSLSSVVTVPSSSLGTKTYTCN PSDEQLKSGTASVVCLL VDHKPSNTKVDKRVESKYGPPCPPCP NNFYPREAKVQWKVDNA APEAAGGPSVFLFPPKPKDTLMISRT LQSGNSQESVTEQDSKD PEVTCVVVDVSQEDPEVQFNWYVDGV STYSLSSTLTLSKADYE EVHNAKTKPREEQFNSTYRVVSVLTV KHKVYACEVTHQGLSSP LHQDWLNGKEYKCKVSNKGLPSSIEK VTKSFNRGEC TISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK PSMB361 EVQLLESGGGLVQPGGSLRLSCAASG 146 EIVLTQSPATLSLSPGE 150 FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSSYLA GSGGYTNYADSMKSRFTISRDNSKNT WYQQKPGQAPRLLIYDA LYLQMNSLRAEDTAVYYCARDSYDSS SNRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSSASTKGP TDFTLTISSLEPEDFAV SVFPLAPCSRSTSESTAALGCLVKDY YYCQQRGNWPLTFGQGT FPEPVTVSWNSGALTSGVHTFPAVLQ KVEIKRTVAAPSVFIFP SSGLYSLSSVVTVPSSSLGTKTYTCN PSDEQLKSGTASVVCLL VDHKPSNTKVDKRVESKYGPPCPPCP NNFYPREAKVQWKVDNA APEAAGGPSVFLFPPKPKDTLMISRT LQSGNSQESVTEQDSKD PEVTCVVVDVSQEDPEVQFNWYVDGV STYSLSSTLTLSKADYE EVHNAKTKPREEQFNSTYRVVSVLTV KHKVYACEVTHQGLSSP LHQDWLNGKEYKCKVSNKGLPSSIEK VTKSFNRGEC TISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK PSMB362 EVQLLESGGGLVQPGGSLRLSCAASG 146 EIVLTQSPATLSLSPGE 148 FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSNYLA GSGGYTNYADSMKSRFTISRDNSKNT WYQQKPGQAPRLLIHDA LYLQMNSLRAEDTAVYYCARDSYDSS SNRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSSASTKGP TDFTLTISSLEPEDFAV SVFPLAPCSRSTSESTAALGCLVKDY YYCQQRRNWPLTFGQGT FPEPVTVSWNSGALTSGVHTFPAVLQ KVEIKRTVAAPSVFIFP SSGLYSLSSVVTVPSSSLGTKTYTCN PSDEQLKSGTASVVCLL VDHKPSNTKVDKRVESKYGPPCPPCP NNFYPREAKVQWKVDNA APEAAGGPSVFLFPPKPKDTLMISRT LQSGNSQESVTEQDSKD PEVTCVVVDVSQEDPEVQFNWYVDGV STYSLSSTLTLSKADYE EVHNAKTKPREEQFNSTYRVVSVLTV KHKVYACEVTHQGLSSP LHQPWLNGKEYKCKVSNKGLPSSIEK VTKSFNRGEC TISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK PSMB363 EVQLLESGGGLVQPGGSLRLSCAASG 146 EIVLTQSPATLSLSPGE 143 FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSSYLA GSGGYTNYADSMKSRFTISRDNSKNT WYQQKPGQAPRLLIYDA LYLQMNSLRAEDTAVYYCARDSYDSS SYRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSSASTKGP TDFTLTISSLEPEDFAV SVFPLAPCSRSTSESTAALGCLVKDY YYCQQRRNWPLTFGQGT FPEPVTVSWNSGALTSGVHTFPAVLQ KVEIKRTVAAPSVFIFP SSGLYSLSSVVTVPSSSLGTKTYTCN PSDEQLKSGTASVVCLL VDHKPSNTKVDKRVESKYGPPCPPCP NNFYPREAKVQWKVDNA APEAAGGPSVFLFPPKPKDTLMISRT LQSGNSQESVTEQDSKD PEVTCVVVDVSQEDPEVQFNWYVDGV STYSLSSTLTLSKADYE EVHNAKTKPREEQFNSTYRVVSVLTV KHKVYACEVTHQGLSSP LHQDWLNGKEYKCKVSNKGLPSSIEK VTKSFNRGEC TISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK PSMB365 EVQLLESGGGLVQPGGSLRLSCAASG 151 EIVLTQSPATLSLSPGE 149 FTFKSDAMHWVRQAPGKGLEWVSEIS RATLSCRASQSVSSYLA GSGGYTNYADSLKSRFTISRDNSKNT WYQQKPGQAPRLLIYDA LYLQMNSLRAEDTAVYYCARDSYDSS SYRATGIPARFSGSGSG LYVGDYFDYWGQGTLVTVSSASTKGP TDFTLTISSLEPEDFAV SVFPLAPCSRSTSESTAALGCLVKDY YYCQQRRNWPLTFGQGT FPEPVTVSWNSGALTSGVHTFPAVLQ KVEIKRTVAAPSVFIFP SSGLYSLSSVVTVPSSSLGTKTYTCN PSDEQLKSGTASVVCLL VDHKPSNTKVDKRVESKYGPPCPPCP NNFYPREAKVQWKVDNA APEAAGGPSVFLFPPKPKDTLMISRT LQSGNSQESVTEQDSKD PEVTCVVVDVSQEDPEVQFNWYVDGV STYSLSSTLTLSKADYE EVHNAKTKPREEQFNSTYRVVSVLTV KHKVYACEVTHQGLSSP LHQDWLNGKEYKCKVSNKGLPSSIEK VTKSFNRGEC TISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK Table 29 provides the CDRs for all for the affinity-matured hits.

TABLE 29 CDR sequences of affinity-matured PSMA hits CDRs (SEQ ID NO:) MAB ID CDR1 CDR2 CDR3 PSMB344 HC SDAMH EISGSGGYTNYADSMKG DSYDSSLYVGDYFDY (25) (130) (27) LC RASQSVSSYLA DASNRAT QQRSNWPLT (28) (29) (30) PSMB345 HC SDAMH EISGSGGYTNYADSMKG DSYDSSLYVGDYFDY (25) (130) (27) LC RASQSVSNYLA DASNRAT QQRRNWPLT (131) (29) (132) PSMB346 HC SDAMH EISGSGGYTNYADSMKG DSYDSSLYVGDYFDY (25) (130) (27) LC RASQSVSSYLA DASYRAT QQRRNWPLT (28) (133) (132) PSMB347 HC SDAMH EISGSGGYTNYADSMKS DSYDSSLYVGDYFDY (25) (134) (27) LC RASQSVSSYLA DASNRAT QQRSNWPLT (28) (29) (30) PSMB349 HC SDAMH EISGSGGYTNYADSLKG DSYDSSLYVGDYFDY (25) (135) (27) LC RASQSVSSYLA DASNRAT QQRGNWPLT (28) (29) (136) PSMB358 HC SDAMH EISGSGGYTNYADSLKG DSYDSSLYVGDYFDY (25) (135) (27) LC RASQSVSSYLA DASNRAT QQRSNWPLT (28) (29) (30) PSMB359 HC SDAMH EISGSGGYTNYADSLKG DSYDSSLYVGDYFDY (25) (135) (27) LC RASQSVSNYLA DASNRAT QQRRNWPLT (131) (29) (132) PSMB360 HC SDAMH EISGSGGYTNYADSLKG DSYDSSLYVGDYFDY (25) (135) (27) LC RASQSVSSYLA DASYRAT QQRRNWPLT (28) (133) (132) PSMB361 HC SDAMH EISGSGGYTNYADSMKS DSYDSSLYVGDYFDY (25) (134) (27) LC RASQSVSSYLA DASNRAT QQRGNWPLT (28) (29) (136) PSMB362 HC SDAMH EISGSGGYTNYADSMKS DSYDSSLYVGDYFDY (25) (134) (27) LC RASQSVSNYLA DASNRAT QQRRNWPLT (131) (29) (132) PSMB363 HC SDAMH EISGSGGYTNYADSMKS DSYDSSLYVGDYFDY (25) (134) (27) LC RASQSVSSYLA DASYRAT QQRRNWPLT (28) (133) (132) PSMB365 HC SDAMH EISGSGGYTNYADSLKS DSYDSSLYVGDYFDY (25) (137) (27) LC RASQSVSSYLA DASYRAT QQRRNWPLT (28) (133) (132)

Example 15. Generation of Affinity-Matured PSMA×CD3 Bispecific

Two types of affinity-matured PSMA×CD3 bispecific antibodies were generated, one specific for the targeting arm (e.g. affinity matured anti-PSMA) recombined with a high affinity CD3 arm [CD3B219 (VH SEQ ID NO: 104, VL SEQ ID NO: 105; HC SEQ ID NO: 110, LC SEQ ID NO: 111)] or a low affinity CD3 arm called CD3B376 [CD3B376 (VH SEQ ID NO: 152, VL SEQ ID NO: 153; HC SEQ ID NO: 154, LC SEQ ID NO: 155)].

TABLE 30 Sequences for the low affinity CD3 arm (CD3B376) SEQ ID SEQ ID MAB ID VH Amino acid sequence NO VL Amo Acid Sequence NO CD3B376 QVQLQQSGPRLVRPSQTLSLTCAISG 152 QSALTQPASVSGSPGQSITISCT 153 DSVFNNNAAWSWIRQSPSRGLEWL GTSSNIGTYKFVSWYQQHPDKA GRTYYRSKWLYDYAVSVKSRITVNPD PKVLLYEVSKRPSGVSSRFSGSKS TSRNQFTLQLNSVTPEDTALYYCARG GNTASLTISGLQAEDQADYHCV YSSSFDYWGQGTLVTVSS SYAGSGTLLFGGGTKLTVL Heavy Chain amino acid Light Chain amino Acid sequence Sequence MAWVWTLLFLMAAAQSIQAQVQL 154 MARSALLILALLLLGLFSPGAWG 155 QQSGPRLVRPSQTLSLTCAISGDSVF QSALTQPASVSGSPGQSITISCT NNNAAWSWIRQSPSRGLEWLGRTY GTSSNIGTYKFVSWYQQHPDKA YRSKWLYDYAVSVKSRITVNPDTSRN PKVLLYEVSKRPSGVSSRFSGSKS QFTLQLNSVTPEDTALYYCARGYSSSF GNTASLTISGLQAEDQADYHCV DYWGQGTLVTVSSASTKGPSVFPLA SYAGSGTLLFGGGTKLTVLGQPK PCSRSTSESTAALGCLVKDYFPEPVTV AAPSVTLFPPSSEELQANKATLV SWNSGALTSGVHTFPAVLQSSGLYSL CLISDFYPGAVTVAWKADSSPV SSVVTVPSSSLGTKTYTCNVDHKPSN KAGVETTTPSKQSNNKYAASSYL TKVDKRVESKYGPPCPPCPAPEAAG SLTPEQWKSHRSYSCQVTHEGS GPSVFLFPPKPKDTLMISRTPEVTCVV TVEKTVAPTECS VDVSQEDPEVQFNWYVDGVEVHNA KIKPREEQFNSTYRVVSVLIVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFLLYSKLTVDKSR WQEGNVFSCSVMHEALHNHYTQKS LSLSLGK

These parental mAbs are in the GenMab format (Labrijn et al, 2013) where the targeting parent (PSMA) contains the 409R GenMab mutation (native amino acid for IgG4), while the killing parent (CD3) contains the F405L GenMab mutation and R409K mutation. The monospecific anti-CD3 antibody was expressed as IgG4, having Fc substitutions S228P, F234A, L235A, F405L, and R409K (CD3 arm) (numbering according to EU index) in their Fc regions. The targeting parent (PSMA) is on human IgG4 with Fc substitutions S228P, F234A, L235A. The monospecific antibodies were expressed in HEK cell lines under CMV promoters.

The parental PSMA and CD3 antibodies were purified using a protein A column with an elution buffer of 100 mM NaAc pH3.5 and a neutralization buffer of 2.5M Tris, pH 7.2. The neutralized parental mAbs were used to make PSMA×CD3 bispecific antibodies. A portion of parental mabs were further buffer exchanged into D-PBS, pH 7.2 buffer for analytical measurements and assays.

Post purification, controlled Fab-arm exchange was performed to make bispecific antibodies as described in Example 6.

The final bispecific antibodies produced, along with the parental mAbs (i.e. PSMA, CD3, or Null) used in the recombination reactions are listed in Table 27 and 28.

Selected PSMA hits were also paired with a non-killing arm (Null) to create negative controls for testing purposes. For control bispecific antibodies, B2M1, an RSV antibody in the IgG4 PAA format was generated, purified and, combined with either the CD3 arm CD3B219-F405L, R409K to generate CD3B288 (CD3×null) or PSMA arms, PSMB122, PSMB126, PSMB130 to generate PS3B37, PS3B39 and PS3B40 respectively (PSMA×null). These PSMA specific affinity matured Mabs were crossed (as in methods above) to CD3B219 and CD3B376 to generate the bispecific antibodies shown in Table 31.

TABLE 31 Generation of affinity-matured PSMA × CD3 bispecific antibodies generated from affinity matured PSMB127 HC LC HC LC SEQ SEQ SEQ SEQ ID Arm 1 ID: ID: Arm 2 ID: ID: PS3B60 PSMB344 145 89 CD3B219 110 111 PS3B61 PSMB345 145 148 CD3B219 110 111 PS3B62 PSMB346 145 149 CD3B219 110 111 PS3B63 PSMB347 146 89 CD3B219 110 111 PS3B64 PSMB349 147 150 CD3B219 110 111 PS3B70 PSMB358 147 89 CD3B219 110 111 PS3B71 PSMB359 147 148 CD3B219 110 111 PS3B72 PSMB360 147 149 CD3B219 110 111 PS3B73 PSMB361 146 150 CD3B219 110 111 PS3B74 PSMB362 146 148 CD3B219 110 111 PS3B75 PSMB363 146 149 CD3B219 110 111 PS3B76 PSMB358 151 149 CD3B376 154 155 PS3B77 PSMB349 145 89 CD3B376 154 155 PS3B78 PSMB359 145 148 CD3B376 154 155 PS3B79 PSMB360 145 149 CD3B376 154 155 PS3B80 PSMB347 146 89 CD3B376 154 155 PS3B81 PSMB361 147 150 CD3B376 154 155 PS3B82 PSMB362 147 89 CD3B376 154 155 PS3B83 PSMB363 147 148 CD3B376 154 155 PS3B84 PSMB344 147 149 CD3B376 154 155 PS3B85 PSMB345 146 150 CD3B376 154 155 PS3B86 PSMB346 146 148 CD3B376 154 155 PS3B90 PSMB365 146 149 CD3B376 154 155

PSMA×CD3 bispecific antibodies were tested for binding to PSMA positive cell line, LNCAP, to PSMA negative cell line, PC3. To assess the binding capabilities of the PSMA bispecific antibodies, the cell-binding assay was utilized (described previously). Bispecific antibodies were normalized for protein concentration and then incubated with the same number of cells expressing either human or cyno PSMA. The MFI at each concentration was collected by flow cytometry and plotted as a function of concentration. Data was transformed via log 10 and then plotted. Nonlinear regression of binding curves was done to determine EC₅₀ s. These relative values were used for ranking PSMA binding to target cells. FIGS. 36-38 show LNCAP binding of all bispecific antibodies prepared. In FIG. 38, none of the constructs demonstrated binding to the PSMA negative cell line. In FIG. 36 and FIG. 37, all of the affinity matured hits demonstrated increased binding affinity through left shifted curves and increased cMax as compared to the parental Mab, PS2B27.

The interactions of the affinity matured bispecific antibodies with recombinant Cyno PSMA ECD and Human PSMA ECD were studied by Surface Plasmon Resonance (SPR) using a ProteOn XPR36 system (BioRad) as described previously for recombinant chimp PSMA ECD. All of the bispecific antibodies bind both targets with substantially the same affinity, KDs ranging from 0.05 nM to 0.27 nM for human PSMA ECD and from 0.05 nM to 0.23 nM from cyno PSMA ECD.

Example 16. Evaluation of PSMA×CD3 Affinity Matured Bispecific Abs in Functional Cell Killing Assay

Based on the above data, affinity measurements and sequence identity, three PSMA antibodies, PSMB347, PSMB360 and PSMB365 as bispecifics with either CD3B219 or CD3B376, were further characterized for the ability to mediate PSMA specific, redirected T cell cytotoxicity. T-cell mediated killing was measured using a caspase cytotoxicity assay, which indirectly measures cell killing via cleavage of a fluorescent substrate by active caspase 3/7. Cleavage of the substrate results in a fluorescent DNA dye, with fluorescence restricted to the cell nucleus. Repeated fluorescence measurements are taken in each well throughout the course of the assay, using a motorized 10× objective, capable of precisely imaging well(s) at the same coordinates. Target cell populations are identified based on defined size restrictions and/or through the use of a secondary label. Frozen Pan CD3+ T-cells (purchased from Biological Specialty Corporation, Colmar, Pa.) were isolated by negative selection from normal healthy donors. Prostate cancer cells, expressing PSMA, (LNCaP, C42) were cultured in RPMI 1640 with 10% HI FBS+supplements (purchased from Life Technologies).

T-cells and target cells were combined at an effector to target ratio (E:T) of 3:1 in Phenol Red free RPMI+10% FBS and supplements (Life Technologies), without selection reagents, and 0.6 uL of NucView caspase reagent (Essen Bioscience) was added to each mL of cells, per manufacturer guidelines. A total volume of 0.1 mL cells were added to appropriate wells of a clear, 96-well flat-bottom plate (BD Falcon). PS3B27 (CD3×PSMA), CD3B288 (CD3×Null) or PS3B46 (PSMAxNull) Bispecific antibodies were prepared at 2× final concentration in Phenol Red free RPMI, prepared as indicated above, and 0.1 mL of compounds were added to each well. After 30 minute incubation at room temperature to minimize cell aggregation at the edge of wells, plates were transferred to the Zoom Incucyte instrument (Essen Bioscience). The Incucyte Instrument resides in a humidified incubator set at 37° C., 5% CO2.

Processing definitions on the Incucyte were designed for each cell line tested, per manufacture guidelines. Measurements were taken every six hours, until a plateau in the caspase signal was observed, and followed by three or more successive decreases from the maximum signal in the well(s) containing the highest concentration of the test compound(s). As the data shows in FIG. 39, the curves for PS3B80, PS3B79, PS3B89, PS3B90, PS3B63, and PS3B72 are left shifted indicating increased potency over PS3B27. The null arm controls did not induce cell death as expected.

Example 17. Anti-Tumor Efficacy in Tumorigenesis Prevention of LnCaP Xenografts in Humanized NSG Mice

Efficacy of PS3B79 and PS3B90 was evaluated in established 3D LnCaP AR.TB human prostate cancer xenografts in male NOD.Cg-Prkdc^(scid)Il2rg^(m)Wjl/SzJ (NSG) mice humanized intraperitoneally (ip) with human T cells. PS3B79 and PS3B90 at 2.5 and 5 mg/kg or Null×CD3B376 antibody control was dosed q3d-q4d on Days 36, 39, 43, 47, 50, 53, 56, 60, and 63 for a total of 8 doses. On day 53 post-tumor implant, which was the last date of the study when nine (9) animals remained per group, tumor growth inhibition (% TGI) was calculated. Statistically significant tumor growth inhibition was observed for PS3B79 at 5 mg/kg with 42% TGI (Two-way ANOVA with Bonferroni test, *p<0.0001, FIG. 40), and for PS3B90 at 2.5 and 5 mg/kg with 53% and 33% respectively compared to Null×CD3 control (Two-way ANOVA with Bonferroni test, *p<0.001, FIG. 41). Thus, CD3B376 is able to induce T cell activation and cytotoxicity in vivo and result in tumor growth inhibition in a bispecific format with high affinity PSMA binding arms, PSMB360 and PSMB365.

Example 18. Efficacy of PSMA×CD3 in the Established Patient-Derived Xenograft LuCaP 86.2 Prostate Tumor Model in T-Cell Humanized NSG Mice

The Antitumor Efficacy of PS3B72 was evaluated in the established patient-derived xenograft (PDX) LuCaP 86.2 prostate tumor model in male NOD.Cg-Prkdc^(scid) Il2rg^(tmlWjt)/SzJ (NSG) mice humanized intraperitoneally (ip) with human pan-T cells. PS3B72 at 0.5 and 5 mg/kg or NullxCD3 antibody control was dosed q3d-q4d on Days 45, 49, 52, 56, 59, 63, 66, 70, 73, and 77 post-tumor implantation for a total of 10 doses. On day 83 post-tumor implant, which was the last day of the study when all ten animals remained per group, tumor growth inhibition (% TGI) was calculated. Statistically significant tumor growth inhibition was observed for PSMA×CD3 at both 0.5 and 5 mg/kg, with 108% and 101% ΔTGI (linear mixed-effect analysis using FDR adjustment, *p<0.0001, FIG. 42), respectively compared to the NullxCD3 control. By the end of the 5 week-dosing period, 9 of 10 complete regressions (CR) were observed in the PSMA×CD3 at 0.5 mg/kg group. These 9 mice remained tumor-free until study termination. At study termination 4 of 10 CR were observed in the PS3B72 at 5 mg/kg group. When mice were dosed with PS3B72 at 5 mg/kg in the absence of T cell humanization, no antitumor efficacy was observed. 

What is claimed: 1) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, wherein the antibody or antigen binding fragment (i) binds to cells expressing recombinant Pan troglodytes PSMA, wherein the binding to cells is measured by flow cytometry and (ii) binds recombinant Pan troglodytes PSMA extracellular domain (SEQ ID NO:4) with an affinity of about 30 nM or less, wherein the affinity is measured by Proteon XPR36 surface plasmon resonance assay. 2) The antibody of claim 1, wherein the antibody has one, two, three or four of the following properties: e) binds LNCaP cells with a calculated EC₅₀ of 20 nM or less and binds Macaca fascicularis PSMA-expressing HEK cells with a calculated EC₅₀ of 40 nM or less, wherein the difference in calculated EC₅₀ between binding LNCaP cells and binding Macaca fascicularis PSMA-expressing HEK cells is less than 5-fold, and wherein the calculated EC₅₀ is measured in a whole cell binding assay at 0° C. using flow cytometry, f) binds recombinant PSMA ECD from human (SEQ ID NO: 7), Pan troglodytes (SEQ ID NO:4) and Macaca fascicularis (SEQ ID NO: 5) with an equilibrium dissociation constant (K_(D)) of 12 nM or less, wherein the K_(D) is measured using Proteon surface plasmon resonance assay ProteOn XPR36 system at +25° C.; g) displays T-cell mediated killing of LNCaP cells, C42 cells, human PSMA-expressing HEK cells or Macaca fascicularis PSMA-expressing HEK cells when paired in a bispecific antibody with anti-CD3 antibody CD3B219, wherein the T-cell mediated killing is measured by Chromium-51 or by caspase 3/7 activation assay or h) recognizes a conformational epitope wherein the epitope is comprised of residues 1138, F235, P237, G238, D244, Y299, Y300, Q303, K304, E307, and K324-P326 of human PSMA (SEQ ID NO:3) 3) The antibody of claim 2, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 14, 15, 16, 17, 18 and 19, respectively. 4) The antibody of claim 2, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 20, 21, 22, 23, 12 and 24, respectively. 5) The antibody of claim 2, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively. 6) The antibody of claim 2, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 38, 39, 40 and 41, respectively. 7) The antibody of claim 2, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 42, 43, 11, 12 and 13, respectively. 8) The antibody of claim 2, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 44, 45, 46, 29 and 47, respectively. 9) The antibody of claim 2, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 48, 49, 50 and 51, respectively. 10) The antibody of claim 2, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 52, 49, 50 and 51, respectively. 11) The antibody of claim 2, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 8, 9, 10, 11, 12 and 13, respectively. 12) The antibody of claim 2, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 32, 33, 34, 12 and 35, respectively. 13) The antibody of claim 2, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 53, 54, 55, 23, 12 and 35, respectively. 14) The antibody of claim 2, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 122, 123, 124, 23, 12 and 24, respectively. 15) The antibody of claim 2, comprising a heavy chain variable region (VH) of SEQ ID NO: 60, 62, 64, 66, 68, 70, 72, 74, 75, 77, 79, or
 160. 16) The antibody of claim 15, comprising a light chain variable region (VL) of SEQ ID NOs: 61, 63, 65, 67, 69, 71, 73, 61, 76, or
 78. 17) The antibody of claim 15, comprising the VH of SEQ ID NO: 62 and the VL of SEQ ID NO:
 63. 18) The antibody of claim 15, comprising the VH of SEQ ID NO: 64 and the VL of SEQ ID NO:
 65. 19) The antibody of claim 15, comprising the VH of SEQ ID NO: 66 and the VL of SEQ ID NO:
 67. 20) The antibody of claim 15, comprising the VH of SEQ ID NO: 72 and the VL of SEQ ID NO:
 73. 21) The antibody of claim 15, comprising the VH of SEQ ID NO: 74 and the VL of SEQ ID NO:
 61. 22) The antibody of claim 15, comprising the VH of SEQ ID NO: 75 and the VL of SEQ ID NO:
 76. 23) The antibody of claim 15, comprising the VH of SEQ ID NO: 77 and the VL of SEQ ID NO:
 78. 24) The antibody of claim 15, comprising the VH of SEQ ID NO: 79 and the VL of SEQ ID NO:
 78. 25) The antibody of claim 15, comprising the VH of SEQ ID NO: 160 and the VL of SEQ ID NO:
 65. 26) The antibody of claim 15, comprising the VH of SEQ ID NO: 60 and the VL of SEQ ID NO:
 61. 27) The antibody of claim 15, comprising the VH of SEQ ID NO: 68 and the VL of SEQ ID NO:
 69. The antibody of claim 15, comprising the VH of SEQ ID NO: 70 and the VL of SEQ ID NO:
 71. 28) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 42, 43, 11, 12 and 13, respectively. 29) The antibody of claim 28, wherein the antibody comprises the VH of SEQ ID NO: 74 and the VL of SEQ ID NO:
 61. 30) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 14, 15, 16, 17, 18 and 19, respectively. 31) The antibody of claim 30, wherein the antibody comprises the VH of SEQ ID NO: 62 and the VL of SEQ ID NO:
 63. 32) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively. 33) The antibody of claim 32, wherein the antibody comprises the VH of SEQ ID NO: 66 and the VL of SEQ ID NO:
 67. 34) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 38, 39, 40 and 41, respectively 35) The antibody of claim 34, wherein the antibody comprises the VH of SEQ ID NO: 72 and the VL of SEQ ID NO:
 73. 36) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 122, 123, 124, 23, 12, and 24, respectively. 37) The antibody of claim 36, wherein the antibody comprises the VH of SEQ ID NO: 160 and the VL of SEQ ID NO:
 65. 38) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 8, 9, 10, 11, 12, and 13, respectively. 39) The antibody of claim 38, wherein the antibody comprises the VH of SEQ ID NO:60 and the VL of SEQ ID NO:61. 40) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 20, 21, 22, 23, 12, and 24, respectively. 41) The antibody of claim 40, wherein the antibody comprises the VH of SEQ ID NO:64 and the VL of SEQ ID NO:65. 42) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 32, 33, 34, 12, and 35, respectively. 43) The antibody of claim 42, wherein the antibody comprises the VH of SEQ ID NO:70 and the VL of SEQ ID NO:71. 44) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 44, 45, 46, 29, and 47, respectively. 45) The antibody of claim 44, wherein the antibody comprises the VH of SEQ ID NO:75 and the VL of SEQ ID NO:76. 46) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 48, 49, 50, and 51, respectively. 47) The antibody of claim 46, wherein the antibody comprises the VH of SEQ ID NO:77 and the VL of SEQ ID NO:78. 48) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 52, 49, 50, and 51, respectively. 49) The antibody of claim 48, wherein the antibody comprises the VH of SEQ ID NO:79 and the VL of SEQ ID NO:78. 50) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 53, 54, 55, 23, 12, and 35, respectively. 51) The antibody of claim 50, wherein the antibody comprises the VH of SEQ ID NO:68 and the VL of SEQ ID NO:69. 52) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 29, and 30, respectively. 53) The antibody of claim 52, wherein the antibody comprises the VH of SEQ ID NO: 138 and the VL of SEQ ID NO:67. 54) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 131, 29, and 132, respectively. 55) The antibody of claim 54, wherein the antibody comprises the VH of SEQ ID NO: 138 and the VL of SEQ ID NO:142. 56) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 133, and 132, respectively. 57) The antibody of claim 56, wherein the antibody comprises the VH of SEQ ID NO:138 and the VL of SEQ ID NO:143. 58) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29, and 30, respectively. 59) The antibody of claim 58, wherein the antibody comprises the VH of SEQ ID NO:139 and the VL of SEQ ID NO:167. 60) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29, and 136, respectively. 61) The antibody of claim 60, wherein the antibody comprises the VH of SEQ ID NO: 140 and the VL of SEQ ID NO:144. 62) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29, and 30, respectively. 63) The antibody of claim 62, wherein the antibody comprises the VH of SEQ ID NO: 140 and the VL of SEQ ID NO:167. 64) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 131, 29, and 132, respectively. 65) The antibody of claim 64, wherein the antibody comprises the VH of SEQ ID NO: 140 and the VL of SEQ ID NO:
 142. 66) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 133, and 132, respectively. 67) The antibody of claim 66, wherein the antibody comprises the VH of SEQ ID NO: 140 and the VL of SEQ ID NO:143. 68) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29, and 136, respectively. 69) The antibody of claim 68, wherein the antibody comprises the VH of SEQ ID NO:139 and the VL of SEQ ID NO:144. 70) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 131, 29, and 132, respectively. 71) The antibody of claim 70, wherein the antibody comprises the VH of SEQ ID NO:139 and the VL of SEQ ID NO:142. 72) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 133, and 132, respectively. 73) The antibody of claim 72, wherein the antibody comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO:143. 74) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 137, 27, 28, 133, and 132, respectively. 75) The antibody of claim 74, wherein the antibody comprises the VH of SEQ ID NO: 141 and the VL of SEQ ID NO:
 143. 76) The antibody of any one of claim 1, wherein the antibody is human or humanized. 77) The antibody of claim 76, wherein the antibody is of IgG4 or IgG1 isotype. 78) The antibody of claim 77, comprising one, two, three, four, five, six, seven, eight, nine or ten substitutions in the antibody Fc. 79) The antibody of claim 77, comprising a) L234A, L235A, G237A, P238S, H268A, A330S and P331S substitutions; b) V234A, G237A, P238S, H268A, V309L, A330S and P331S substitutions; c) F234A, L235A, G237A, P238S and Q268A substitutions, d) L234A, L235A or L234A and L235A substitutions; e) F234A, L235A or F234A and L235A substitutions; or f) V234A substitution, wherein residue numbering is according to the EU Index. 80) The antibody of claim 79, comprising S228P, F234A and L235A substitutions, wherein residue numbering is according to the EU Index. 81) The antibody of any one of claim 1, wherein the antibody is bispecific. 82) The antibody of claim 76, wherein the antibody specifically binds PSMA and specifically binds CD3, CD5, CD28, CD16, CD16A, CD25, CD38, CD44, CD56, CD69, CD94, CD335 (NKp46), CD336, (NKp44), CD337 (NKp30), NKp80, NKG2C and NKG2D, DNAM, NCRs, CD18, CD89, CD18, CD32, CD64, CD64 and CD35. 83) A pharmaceutical composition comprising the antibody of any one of claim 1 and a pharmaceutically accepted carrier. 84) A polynucleotide encoding the antibody VH of claim 15, the antibody VL of claim 16, or the antibody VH and the antibody VL of claim 15 and
 16. 85) A polynucleotide encoding the antibody VH, the antibody VL, or the antibody VH and the antibody VL of claim
 1. 86) A vector comprising the polynucleotide of claim
 84. 87) A vector comprising the polynucleotide of claim
 85. 88) A host cell comprising the vector of claim
 86. 89) A host cell comprising the vector of claim
 87. 90) A method of producing the antibody of claim 1, comprising culturing the host cell of claim 89 in conditions that the antibody is expressed, and recovering the antibody produced by the host cell. 91) A method of treating a cancer in a subject, comprising administering a therapeutically effective amount of the isolated antibody of any one of claim 1 to the subject in need thereof for a time sufficient to treat the cancer. 92) The method of claim 91, wherein the cancer is a solid tumor, malignancy or a tumor neovasculature. 93) The method of claim 92, wherein the solid tumor is a prostate cancer or a colorectal cancer, a gastric cancer, a clear cell renal carcinoma, a bladder cancer, a lung cancer, a squamous cell carcinoma, a glioma, a breast cancer, a kidney cancer, a neovascular disorder, a clear cell renal carcinoma (CCRCC), a pancreatic cancer, a renal cancer, a urothelial cancer and an adenocarcinaoma to the liver. 94) The method of claim 93, wherein the prostate cancer is a refractory prostate cancer, a prostatic intraepithelial neoplasia, an androgen independent prostate cancer, a malignant prostate cancer. 95) The method of any one of claims 90-94, wherein the antibody is administered in combination with a second therapeutic agent. 96) The method of claim 95, wherein the second therapeutic agent is a standard of care drug for treatment of the solid tumor or malignancy or a tumor neovasculature. 97) The method of claim 96, wherein the second therapeutic agent is an hormone inhibitor, an antimicrotubule agent, a kinase inhibitor, an immunomodulating agent, a topoisomerase inhibitor, an anti-metabolite, a mitotic inhibitor, an alkylating agent, an anthracycline, a vinca alkaloid, an intercalating agent, an agent capable of interfering with a signal transduction pathway, an agent that promotes apoptosis, a proteosome inhibitor or radiation. 98) The method of claim 96, wherein the second therapeutic agent is a vaccine. 99) The method of claim 98, wherein the vaccine is a polypeptide or fragment thereof, or a DNA or a RNA encoding the polypeptide or fragment thereof expressed on tumor cells. 100) The method of claim 99, wherein the polypeptide is PSMA, mesothelin, EGFR or EGFRvIII. 101) The method of claim 95, wherein the second therapeutic agent is administered simultaneously, sequentially or separately. 102) The method of any one of claims 91-101, wherein the subject is treated or is being treated with radiation therapy. 103) The method of any one of claims 91-101 wherein the subject has had or will undergo surgery. 104) The method of any one of claims 46-58, wherein the isolated antibody comprises the VH of SEQ ID NO: 66 and the VL of SEQ ID NO:
 67. 105) The antibody of any one of claim 1 for use in therapy. 106) An anti-idiotypic antibody binding to the antibody of any one of claim
 1. 107) A bispecific antibody comprising a first domain that specifically binds PSMA and a second domain that specifically binds CD3, wherein the first domain comprises: a) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 42, 43, 11, 12 and 13, respectively; b) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 14, 15, 16, 17, 18 and 19, respectively; c) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively; d) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 38, 39, 40 and 41, respectively; e) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 122, 123, 124, 23, 12, and 24, respectively; f) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 8, 9, 10, 11, 12, and 13, respectively; g) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 14, 15, 16, 17, 18, and 19, respectively; h) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 20, 21, 22, 23, 12, and 24, respectively; i) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 32, 33, 34, 12, and 35, respectively; j) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 44, 45, 46, 29, and 47, respectively; k) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 48, 49, 50, and 51, respectively; l) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 53, 54, 55, 23, 12, and 35, respectively; m) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 29, and 30, respectively; n) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 131, 29, and 132, respectively; o) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 133, and 132, respectively; p) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29, and 30, respectively; q) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29, and 136, respectively; r) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29, and 30, respectively; s) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 131, 29, and 132, respectively; t) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 133, and 132, respectively; u) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29, and 136, respectively; v) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 131, 29, and 132, respectively; w) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 133, and 132, respectively; or x) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 137, 27, 28, 133, and 132, respectively. 108) The bispecific antibody of claim 107, wherein the first domain comprises: a) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 42, 43, 11, 12 and 13, respectively, and the VH of SEQ ID NO: 74 and the VL of SEQ ID NO: 61; b) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 14, 15, 16, 17, 18 and 19, respectively, and the VH of SEQ ID NO: 62 and the VL of SEQ ID NO: 63; c) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively, and the VH of SEQ ID NO: 66 and the VL of SEQ ID NO: 67; d) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 38, 39, 40 and 41, respectively, and the VH of SEQ ID NO: 72 and the VL of SEQ ID NO: 73; e) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 122, 123, 124, 23, 12, and 24, respectively, and the VH of SEQ ID NO: 160 and the VL of SEQ ID NO: 65; f) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 8, 9, 10, 11, 12, and 13, respectively, and the VH of SEQ ID NO:60 and the VL of SEQ ID NO:61; g) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 14, 15, 16, 17, 18, and 19, respectively, and the VH of SEQ ID NO:62 and the VL of SEQ ID NO:63; h) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 20, 21, 22, 23, 12, and 24, respectively, and VH of SEQ ID NO:64 and the VL of SEQ ID NO:65; i) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 32, 33, 34, 12, and 35, respectively, and the VH of SEQ ID NO:70 and the VL of SEQ ID NO:71; j) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 31, 44, 45, 46, 29, and 47, respectively, the VH of SEQ ID NO:75 and the VL of SEQ ID NO:76; k) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 37, 48, 49, 50, and 51, respectively, and the VH of SEQ ID NO:77 and the VL of SEQ ID NO:78; l) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 53, 54, 55, 23, 12, and 35, respectively, and the VH of SEQ ID NO:68 and the VL of SEQ ID NO:69; m) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 29, and 30, respectively, and the VH of SEQ ID NO:138 and the VL of SEQ ID NO:67; n) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 131, 29, and 132, respectively, and the VH of SEQ ID NO:138 and the VL of SEQ ID NO: 142; o) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 130, 27, 28, 133, and 132, respectively, and the VH of SEQ ID NO:138 and the VL of SEQ ID NO:143; p) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29, and 30, respectively, and the VH of SEQ ID NO:139 and the VL of SEQ ID NO:167; q) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29, and 136, respectively, and the VH of SEQ ID NO:140 and the VL of SEQ ID NO:144; r) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 29, and 30, respectively, and the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 167; s) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 131, 29, and 132, respectively and the VH of SEQ ID NO:140 and the VL of SEQ ID NO: 142; t) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 135, 27, 28, 133, and 132, respectively, and the VH of SEQ ID NO:140 and the VL of SEQ ID NO:143; u) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 29, and 136, respectively; and the VH of SEQ ID NO:139 and the VL of SEQ ID NO: 144; v) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 131, 29, and 132, respectively, and the VH of SEQ ID NO:139 and the VL of SEQ ID NO: 142; w) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 134, 27, 28, 133, and 132, respectively, and the VH of SEQ ID NO:139 and the VL of SEQ ID NO:143; or x) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 25, 137, 27, 28, 133, and 132, respectively and the VH of SEQ ID NO:141 and the VL of SEQ ID NO:
 143. 109) An isolated bispecific PSMA/CD3 antibody comprising a first domain that (i) binds to cells expressing recombinant Pan troglodytes PSMA, wherein the binding to cells is measured by flow cytometry and (ii) binds recombinant Pan troglodytes PSMA extracellular domain (SEQ ID NO:4) with an affinity of about 30 nM or less, wherein the affinity is measured by Proteon surface plasmon resonance assay specifically binding PSMA, and a second domain specifically binding CD3. 110) The bispecific PSMA×CD3 antibody of claim 109, wherein the antibody e) binds LNCaP cells with a calculated EC₅₀ of 20 nM or less and binds Macaca fascicularis PSMA-expressing HEK cells with a calculated EC₅₀ of 40 nM or less, wherein the difference in calculated EC₅₀ between binding LNCaP cells and binding Macaca fascicularis PSMA-expressing HEK cells is less than 5-fold, and wherein the calculated EC₅₀ is measured in a whole cell binding assay at 0° C. using flow cytometry, f) binds recombinant PSMA ECD from human (SEQ ID NO:7), Pan troglodytes (SEQ ID NO:4) and Macaca fascicularis (SEQ ID NO:5) with an equilibrium dissociation constant (K_(D)) of 12 nM or less, wherein the K_(D) is measured using Proteon surface plasmon resonance assay ProteOn XPR36 system at +25C; g) displays T-cell mediated killing of LNCaP cells, C42 cells, human PSMA-expressing HEK cells or Macaca fascicularis PSMA-expressing HEK cells, wherein the T-cell mediated killing is measured by Chromium-51 or by caspase 3/7 activation assay or h) recognizes a conformational epitope wherein the epitope is comprised of residues I1138, F235, P237, G238, D244, Y299, Y300, Q303, K304, E307, and K324-P326 of human PSMA (SEQ ID NO:3). 111) The bispecific PSMA×CD3 antibody of claim 109, wherein the antibody binds to T-cells. 112) The bispecific PSMA×CD3 antibody of claim 109, wherein the first domain comprises g) the heavy chain complementarity determining region 1 (HCDR1), a HCDR2 and a HCDR3 of SEQ ID NOs: 14, 15 and 16, respectively; and the light chain complementarity determining region 1 (LCDR1), a LCDR2 and a LCDR3 of SEQ ID NOs: 17, 18 and 19, respectively; h) the HCDR1, the HCDR2 and the HCDR3 of SEQ ID NOs: 20, 21 and 22, respectively, and the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 23, 12 and 24, respectively; i) the HCDR1, the HCDR2 and the HCDR3 of SEQ ID NOs: 25, 26 and 27, respectively, and the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 28, 29 and 30, respectively; j) the HCDR1, the HCDR2 and the HCDR3 of SEQ ID NOs: 31, 44 and 45, respectively, and the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 46, 29 and 47, respectively; k) the HCDR1, the HCDR2 and the HCDR3 of SEQ ID NOs: 31, 42 and 43, respectively, and the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 11, 12 and 13, respectively; or l) the HCDR1, the HCDR2 and the HCDR3 of SEQ ID NOs: 122, 123 and 124, respectively, and the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 23, 12 and 24, respectively. 113) The bispecific PSMA×CD3 antibody of claim 109, wherein the first domain comprises the HCDR1, the HCDR2 and the HCDR3 of g) SEQ ID NOs: 14, 15 and 16, respectively; h) SEQ ID NOs: 20, 21 and 22, respectively; i) SEQ ID NOs: 25, 26 and 27, respectively, j) SEQ ID NOs: 31, 44 and 45, respectively; k) SEQ ID NOs: 31, 42 and 43, respectively; or l) SEQ ID NOs: 122, 123 and 124, respectively. 114) The bispecific PSMA×CD3 antibody of claim 109, wherein the first domain comprises the LCDR1, the LCDR2 and the LCDR3 of g) SEQ ID NOs: 17, 18 and 19, respectively; h) SEQ ID NOs: 23, 12 and 24, respectively; i) SEQ ID NOs: 28, 29 and 30, respectively; j) SEQ ID NOs: 46, 29 and 47, respectively; k) SEQ ID NOs: 11, 12 and 13, respectively; or l) SEQ ID NOs: 23, 12 and 24, respectively. 115) The bispecific PSMA×CD3 antibody of claim 109, wherein g) the first domain comprises a heavy chain variable region (VH) of SEQ ID NO: 62 and a light chain variable region (VL) of SEQ ID NO: 63, and the second domain comprises the VH of SEQ ID NO: 104 and the VL of SEQ ID NO: 105 h) the first domain comprises the VH of SEQ ID NO: 64 and the VL of SEQ ID NO: 65, and the second domain comprises the VH of SEQ ID NO: 104 and the VL of SEQ ID NO: 105; i) the first domain comprises the VH of SEQ ID NO: 66 and the VL of SEQ ID NO: 67, and the second domain comprises the VH of SEQ ID NO: 104 and the VL of SEQ ID NO: 105; j) the first domain comprises the VH of SEQ ID NO: 75 and the VL of SEQ ID NO: 76, and the second domain comprises the VH of SEQ ID NO: 104 and the VL of SEQ ID NO: 105; k) the first domain comprises the VH of SEQ ID NO: 74 and the VL of SEQ ID NO: 61, and the second domain comprises the VH of SEQ ID NO: 104 and the VL of SEQ ID NO: 105; l) the first domain comprises the VH of SEQ ID NO: 160 and the VL of SEQ ID NO: 65, and the second domain comprises the VH of SEQ ID NO: 104 and the VL of SEQ ID NO:
 105. 116) The bispecific PSMA×CD3 antibody of claim 109, comprising a first heavy chain (HC1), a first light chain (LC1), a second heavy chain (HC2) and a second light chain (LC2), wherein the HC1 and the LC1 comprise the amino acid sequences of g) SEQ ID NOs: 84 and 85, respectively; h) SEQ ID NOs: 86 and 87, respectively; i) SEQ ID NOs: 88 and 89, respectively; j) SEQ ID NOs: 125 and 91, respectively; k) SEQ ID NOs: 94 and 95, respectively; or l) SEQ ID NOs: 96 and 83, respectively. 117) The bispecific PSMA×CD3 antibody of claim 116, wherein the HC2 and the LC2 comprises SEQ ID NOs: 110 and 111, respectively. 118) The bispecific PSMA×CD3 antibody of claim 109, comprising the HC1, the LC1, the HC2 and the LC2 of g) SEQ ID NOs: 84, 85, 110 and 111, respectively; h) SEQ ID NOs: 86, 87, 110 and 111, respectively; i) SEQ ID NOs: 88, 89, 110, 111, respectively; j) SEQ ID NOs: 125, 91, 110 and 111, respectively; k) SEQ ID NOs: 94, 95, 110 and 111, respectively; l) SEQ ID NOs: 96, 83, 110 and 111, respectively. 119) The bispecific PSMA×CD3 antibody of any one of claim 109, wherein the antibody is human or humanized. 120) The bispecific PSMA×CD3 antibody of claim 119, wherein the antibody is of IgG1, IgG2, IgG3 or IgG4 isotype. 121) The bispecific PSMA×CD3 antibody of claim 120, wherein the antibody is of IgG1 or IgG4 isotype. 122) The bispecific PSMA×CD3 antibody of claim 120 or 121, having one, two, three, four, five, six, seven, eight, nine or ten substitutions in an antibody Fc. 123) The bispecific PSMA×CD3 antibody of claim 121, comprising: a) L234A, L235A, G237A, P238S, H268A, A330S and P331 S substitutions; b) V234A, G237A, P238S, H268A, V309L, A330S and P331S substitutions; c) F234A, L235A, G237A, P238S and Q268A substitutions; d) L234A, L235A or L234A and L235A substitutions; e) F234A, L235A or F234A and L235A substitutions; f) V234A substitution, or g) S228P, F234A and L235A substitutions, wherein residue numbering is according to the EU Index. 124) The bispecific PSMA×CD3 antibody of any one of claim 109, comprising at least one substitution in an antibody CH3 constant domain. 125) The bispecific PSMA×CD3 antibody of claim 124, wherein the substitution in the antibody CH3 constant domain is 409R, F405L or F405L/R409K substitution, wherein residue numbering is according to the EU Index. 126) The bispecific PSMA×CD3 antibody of claim 124, wherein the antibody comprises a) F405L substitution in the HC1 and 409R substitution in the HC2, wherein the antibody is of IgG1 isotype; b) V234A, G237A, P238S, H268A, V309L, A330S, P331S and F405L substitutions in the HC1 and V234A, G237A, P238S, H268A, V309L, A330S, P331S and 409R substitutions in the HC2, wherein the antibody is of IgG1 isotype; or b) S228P substitution in the HC1 and S228P, F405L and R409K substitution in the HC2, wherein the antibody is of IgG4 isotype. 127) A pharmaceutical composition comprising the bispecific PSMA×CD3 antibody of any one of claim 109 and a pharmaceutically accepted carrier. 128) A polynucleotide encoding the bispecific PSMA×CD3 antibody HC1, LC1, HC2 or LC2 of claim
 118. 129) A vector comprising the polynucleotide encoding the HC1, the LC1, the HC2, the LC2, the HC1 and the LC1 or the HC2 and the LC2 of claim
 128. 130) An isolated host cell comprising the vector of claim
 129. 131) A method of producing the bispecific PSMA×CD3 antibody of claim 118, comprising culturing the host cell of claim 130 in conditions that the antibody is expressed, and recovering and purifying the bispecific PSMA×CD3 antibody produced by the host cell. 132) A method of producing the bispecific PSMA×CD3 antibody of claim 118, comprising: a) combining a monospecific bivalent PSMA antibody having two identical HC1 and two identical LC1 and a monospecific bivalent CD3 antibody having two identical HC2 and two identical LC2 in a mixture of about 1:1 molar ratio; b) introducing a reducing agent into the mixture; c) incubating the mixture about ninety minutes to about six hours; d) removing the reducing agent; and e) purifying the bispecific PSMA×CD3 antibody that comprises the HC1, the LC1, the HC2 and the LC2. 133) The method of claim 132, wherein the reducing agent is 2-mercaptoethanolamine (2-MEA). 134) The method of claim 133, wherein h) the 2-MEA is present at a concentration of about 25 mM to about 75 mM; and i) the incubating step is performed at a temperature of about 25° C. to about 37° C. 135) A method of treating a cancer in a subject, comprising administering a therapeutically effective amount of the isolated PSMA×CD3 bispecific antibody of any one of claims 109-126 to the subject in need thereof for a time sufficient to treat the cancer. 136) The method of claim 135, wherein the cancer is a solid tumor, malignancy or a tumor neovasculature. 137) The method of claim 136, wherein the solid tumor is a prostate cancer or a colorectal cancer, a gastric cancer, a clear cell renal carcinoma, a bladder cancer, a lung cancer, a squamous cell carcinoma, a glioma, a breast cancer, a kidney cancer, a neovascular disorder, a clear cell renal carcinoma (CCRCC), a pancreatic cancer, a renal cancer, a urothelial cancer and an adenocarcinaoma to the liver. 138) The method of claim 137, wherein the prostate cancer is a refractory prostate cancer, a prostatic intraepithelial neoplasia, an androgen independent prostate cancer, a malignant prostate cancer. 139) The method of any one of claims 135-138, wherein the antibody is administered in combination with a second therapeutic agent. 140) The method of claim 139, wherein the second therapeutic agent is a standard of care drug for treatment of the solid tumor or malignancy or a tumor neovasculature. 141) The method of claim 139, wherein the second therapeutic agent is a hormone inhibitor, an antimicrotubule agent, a topoisomerase inhibitor, an anti-metabolite, a mitotic inhibitor, an alkylating agent, an anthracycline, a vinca alkaloid, an intercalating agent, an agent capable of interfering with a signal transduction pathway, an agent that promotes apoptosis, a proteosome inhibitor or radiation. 142) The method of claim 139, wherein the second therapeutic agent is a vaccine. 143) The method of claim 142, wherein the vaccine is a polypeptide or fragment thereof, or a DNA or a RNA encoding the polypeptide or fragment thereof expressed on tumor cells. 144) The method of claim 143, wherein the polypeptide is PSMA, mesothelin, EGFR or EGFRvIII. 145) The method of claim 139, wherein the second therapeutic agent is administered simultaneously, sequentially or separately. 146) The method of any one of claims 135-145, wherein the subject is treated or is being treated with radiation therapy. 147) The method of any one of claims 135-145 wherein the subject has had or will undergo surgery. 148) The method of any one of claims 135-145, wherein the first domain of the bispecific PSMA×CD3 antibody comprises the VH of SEQ ID NO:66 and the VL or SEQ ID NO:67, and the second domain of the bispecific PSMA×CD3 antibody comprises the VH of SEQ ID NO:104 and the VL of SEQ ID NO:105. 149) The antibody of any one of claim 109 for use in therapy. 150) An anti-idiotypic antibody binding to the antibody of any one of claim
 109. 